Revision as of 07:41, 13 October 2016

iGEM TU Delft

Notebook

Day Notes

June

29^th June 2016

María

Prepare 10mL of antibiotic stocks of both antibiotics that are going to be used during the project: Chloramphenicol (Cm) and Ampicillin (Amp). Make 500µL and 1mL aliquots.

Prepare 1L of LB medium and 3 400mL bottles of LB Agar. Send to autoclave.

30^th June 2016

María

Add antibiotics to the LB Agar bottles prepared the previous day and pour plates to have them ready for when we start transforming.

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July

4^th July 2016

Lycka

Digestion gBlocks mVenus and mKate with EcoRI and PstI.

Made liquid culture of strain containing pSB4A5 bakcbone with RFP.

María

Digestion of pSB1C3 linearized backbone from the distribution kit with EcoRI and PstI.

Charlotte

Made two liquid overnight cultures of E. coli K12 harboring the standard backbone pSB4A5 containing RFP.

5^th July 2016

María

Dissolution of necessary parts from distribution kit: GFP BioBrick BBa_E0840 and promoters BBa_J23100, BBa_J23105, BBa_J23108, BBa_J23113 and BBa_J23117.

Dissolution of K1149051 (a kind gift of Imperial College), which was sent dry on filter paper.

Lycka

Ligation of mKate into backbone pSB1C3.

Transformation of ligation product mKate and the BioBricks dissolved by María into Escherichia coli Top10 cells. These were plated on LB medium supplemented with Cm.

Charlotte

Did 10 minipreps on the overnight culture with RFP pSB4A5, according to protocol.

6^th July 2016

Lycka

Stocked primers VF2 and VR: storage stock (100µM) and working stock (10µM).

Colony PCR of transformants from yesterday with primers VF2 and VR followed by gel electrophoresis. Gel picture yielded no bands. This might be attributed to a fault in the transformation.

7^th July 2016

María

Transformation of same BioBricks as on 5^th July using a different transformation protocol since we believed that the negative result was due to a mistake during transformation.

8^th July 2016

María

Finally, the plasmids containing K1149051 and BBa_E0840 yielded a few colonies. However, the ones containing the promoters did not. Thus, we believe that the distribution kit plate containing the promoters is defective and the mKate ligation also featured some problems.

11^th July 2016

Lycka

To test whether the distribution kit might be the problem, transformation of BBa_J23113 in OneShot® TOP10 chemically competent cells (Invitrogen) to make sure the transformation process is not the cause of the negative results. Also, a plasmid containing TU Delft 2015 csgA Biobrick was used as a positive control.

For the mKate construct we will obtain the backbone from another construct instead of the linearized backbones from the iGem distribution kit.

12^th July 2016

Lycka

Measure DNA concentration of biobricks from registry by nanodrop.

Nanodrop

Product	Concentration (ng/µl)
J23113	89.3
J23117	91.7
J23105	89.3
J23108	92.7
J23100	93.4
E0840	81.9

As can be seen from the table, there is DNA presence in the samples. Therefore, something else should be causing the transformations not to work. Since last year's distribution kit plates are still available in the lab and those plasmids were not taken we will try to get these parts from 2015 distribution kit.

María

Transfer colonies for K1149051 and BBa_E0840 to overnight LB+Cm culture. A colony containing CsgA was transfered as well to be able to isolate and use that plasmid as standard backbone for all synthesized parts.

13^th July 2016

María

Plasmid isolation of overnight cultures prepared yesterday and preparation of K1149051 and BBa_E0840 samples for sequencing.

Lycka

Cryostocks of K1149051 and BBa_E0840 overnight culures. Stored at -80 degrees.

Digestion of backbones pSB1C3, pSB4A5 and all gBlocks with EcorI and PstI.

14^th July 2016

Lycka

Gel purification of digested backbones. Dissolved in nuclease free water, stored at -20 degrees.

Ligation of synthesized fragments into backbones.

María

After all the problems transforming in the past weeks, we tested if we could use our homemade chemically competent cells for future transformations by transforming a couple of positive controls, which were taken from last year's plasmids. The transformations worked so these cells can still be used.

15^th July 2016

María

Transformation of Lycka's ligation products from yesterday and the BioBricks from the distribution kit were given one last chance. Transformed cells were plated after doubling their concentration.

Lycka

Dissolution of promoter BioBricks from 2015 iGEM distribution kit.

18^th July 2016

Lycka

Restriction of backbones pSB1C3 and pSB4A5 with EcorI and PstI. Gel electrophoresis of digested fragments. Gel picture yielded no bands.

19^th July 2016

Lycka

Transformation of BioBricks from the distribution kit of 2015, since the ones from 2016 yielded no colonies. Positive control: csgA. Negative control: water. Biobricks: J23100, J23108, J23105, J23117, J23113. Overnight cultivation yielded no result, we decide to drop this and add the promoters to the parts that need it by PCR.

María

Restriction of pSB1C3 and pSB4A5 backbones with EcoRI and PstI

Ligation of synthesized parts that could not be ligated on 14^th of July: INP_Sil_Sdom in pSB1C3 and BolA_ind and BolA_con in both pSB1C3 and pSB4A5.

20^th July 2016

Lycka and Tessa

Transformation of plasmids ligated by Maria (19^th of July) together with the ones ligated on 14^th of July. After overnight culture the following plates contained colonies. pSB1C3: INP_Sil_Sdom, OmpA_Sil_Taur, Sil_Sdom, SulA, BolA_ind, BolA_con, phaP. pSB4A5: BolA_con. The ones with a negative result were ligated again the next day.

21^st July 2016

María

Colony PCR of yesterday's transformation results from Lycka and Tessa. However, not all transformations yielded colonies.

Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in selective LB overnight.

Lycka

Ligation of mKate, mVenus, mCerulean, OmpA_Sil_Sdom and LacI into pSB1C3. Ligation of OmpA_Sil_Taur, OmpA_Sil_Sdom, Sil_Sdom, SulA and BolA_ind into pSB4A5. Left at room temperature overnight.

22^nd July 2016

María

Continue with colony PCR of 20^th of July transformation results from Lycka and Tessa.

Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in selective LB overnight.

Lycka

Transformation of ligation products from yesterday. Cells containing backbones pSB1C3 or pSB4A5 were plated on plates supplemented with chloramphenicol or ampicilin, respectively. After overnight cultivation the following plates contained colonies: mKate, mVenus, mCerulean, OmpA_Sil_Sdom, Sil_Sdom, SulA, BolA_ind.

23^rd July 2016

María

Continue with colony PCR of yesterday's transformation results from Lycka.

Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in selective LB overnight.

Pick all colonies selected by colony PCR on 21^st, 22^nd and 23^rd of July and transfer to selective LB according to the resistance expressed by each plasmid.

24^th July 2016

Lycka

Cryostock and plasmid isolation of overnight cultures prepared yesterday by Maria. Cryostocks stored at -80 degrees, isolated plasmids stored at -20 degrees.

25^th July 2016

Lycka

Nanodrop and prepare for sequencing plasmids isolated yesterday.

Sequencing resulted in the following. Correct sequence: Sil_Sdom (pSB1C3), OmpA_Sil_Taur (pSB1C3), Sil_Sdom (pSB4A5), SulA (pSB4A5). Sequence with mutations: SulA (pSB1C3), BolA_ind (pSB1C3), J23108 (pSB1C3), BolA_con (pSB4A5), BolA_ind (pSB4A5). Different sequence: BolA_con (pSB1C3), INP_sil_Sdom (pSB1C3), OmpA_Sil_Sdom (pSB4A5). Mutated sequences can be repaired by PCR and blunt end ligation.

María

Prepare new digested pSB1C3 and pSB4A5 backbones by restricting with EcoRI and PstI and gel isolating the restricted product.

New ligation of those synthesized fragments that have not yielded any positive results (transformation or sequence confirmation) in the past week in their respective backbones.

27^th July 2016

Lycka

Colony PCR of plates transformed yesterday.

Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated the same day in selective LB overnight.

María and Tessa

Make new chemically competent cells. They were tested for transformation capacity with a positive control plasmid (RFP in pSB4A5). Also, untransformed cells were plated in plates supplemented with Cm or Amp to test whether they had developed a resistance during the process.

28^th July 2016

María

Cryostock, plasmid isolation and preparation of samples for sequencing of the overnight cultures prepared yesterday by Lycka. Only INP_Sil_Sdom in pSB4A5 was sequenced confirmed with a mutation to be fixed by PCR. The other sequences aligned to a fragment of GFP unknown to us, we assumed it either came from a contamination of the old restriction enzymes we were using or from the pSB1C3 backbone that was taken from last year's team plasmid stock and probably was poorly labeled. Thus, we decided to use another backbone and the new enzymes sponsored to all iGEM teams by New England Biolabs.

29^th July 2016

Lycka and Célina

Repeat of the colony PCR of plates from the 26^th of July that yielded a negative result.

Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in LB and the applicable antibiotic overnight.

Transformation of OmpA_Sil_Sdom in both different backbones.

30^th July 2016

Lycka

Colony PCR of OmpA_Sil_Sdom in both backbones from the 29^th of July. The PCR machine was filled with colonies from older plates which had not yielded any good colonies yet: mKate (26^th of July), mKate (mKate 30^th of June), INP_Sil_Sdom (20^th of July).

Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in LB and the applicable antibiotic overnight.

Cryostocking and plasmid isolation of colonies picked by on 29^th of July. Cryostocks stored at -80 degrees, isolated plasmid stored at -20 degrees.

31^st July 2016

María

Cryostock and plasmid isolation of the overnight cultures prepared yesterday by Lycka and preparation of samples for sequencing of plasmids isolated yesterday and today. Sequence of mCerulean was confirmed (although it had a silent mutation). OmpA_Sil_Sdom in pSB4A5 and INP_Sil_Sdom in pSB1C3 had a deletion to be fixed by PCR. The rest either of sequences contained the random GFP sequenced mentioned before (since they are old ligation products) or multiple mutations that could not be fixed by PCR.

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August

2^nd August 2016

Tessa

Amplified E0840 (GFP) out of a pSB1C3-GFP plasmid using Phusion PCR. Four reactions of 50µl.

Nanodropped PCR products.

Nanodrop

Product	Concentration (ng/µl)
E0840	339.6
E0840	554.1
E0840	596.9
E0840	567.6

Ran a 1% agarose gel of the PCR product to verify product size.

Stored product 2 and 4 in the fridge for later use.

Lara

Digested the synthesized parts LacI, phaP, mKate, the backbones pSB1C3 and pSB4A5 and the Biobrick K1149051 with EcoRI and PstI. The restriction products were run over a 1% agarosegel and subsequently gel-purified.

Concentrations of purified DNA products.

Nanodrop

DNA construct	Concentration (ng/µl)
LacI	15.5
phaP	8.2
mKate	8.7
K1149051	13.8
pSB1C3	25.5
pSB1C3	13.1
pSB1C3	9.3
pSB4A5	10.4
pSB4A5	7.3
pSB4A5	6.4

3^rd August 2016

Lara

Ligated the synthesized parts that were restricted and gel-purified yesterday and the backbones. The following ligation products were formed: LacI-pSB1C3, phaP-pSB1C3, mKate-pSB1C3, LacI-pSB4A5 and K1149051-pSB4A5. The ligation protocol was used and the below stated lengths for the vector and the inserts were used.

Lengths of inserts and backbones.

DNA construct	DNA length (bp)
LacI	1335
phaP	648
mKate	790
K1149051	4271
pSB1C3	2070
pSB4A5	3395

Transformations were performed with the just obtained ligation products and additionally the previously obtained mVenus-pSB1C3 was also transformed into TOP10 cells. A DNA amount of 2 µL was used; as positive control for the ampicilline plates the plasmid RFP-pSB4A5 was used during transformation and for the chloramphenicol plates the plasmid RFP-pSB1C3 was used. Transformed cells were plated on selective LB-agar plates and incubated overnight at 37 degrees Celsius.

4^th August 2016

Lara

Performed a DreamTaq colony PCR for yesterday's transformations; eight colonies per plate were picked and analyzed.

Colonies were transferred into selective liquid LB-medium.

DNA construct transformation	Selective medium	Colonies picked
K1149051-pSB4A5	LB-Amp	2
phaP-pSB1C3	LB-Cm	19, 20 and 21
mKate-pSB1C3	LB-Cm	31
mVenus-pSB1C3	LB-Cm	36, 40 and 41
LacI-pSB1C3	LB-Cm	46, 48 and 49

5^th August 2016

Lara

Some of the overnight cultures were opened inside the shaker, so these were not sterile anymore. Colonies 21 (phaP-pSB1C3) and 47 (LacI-pSB1C3) had to be picked and transferred into liquid LB-medium again.

From the other overnight cultures, plasmids were isolated using Miniprep.

Concentrations of plasmid isolations.

DNA construct	Colony number	Concentration (ng/L)
K1149051-pSB4A5	2	271.7
phaP-pSB1C3	19	398.0
phaP-pSB1C3	20	207.1
mKate-pSB1C3	31	83.3
mVenus-pSB1C3	36	142.5
mVenus-pSB1C3	40	306.5
mVenus-pSB1C3	41	65.3

These DNA samples were sent to sequencing.

9^th August 2016

Charlotte

Started correcting point mutations using PCR in BioBricks that had a non-silent mutation in a coding area. The plasmids being corrected are SulA (pSB1C3), BolA_ind (pSB1C3), BolA_con (pSB4A5) and INP_Sil_Sdom (pSB4A5). Primers were ordered by María. Primers were first diluted for storage (100 µM) and working stock (10 µM). The concentrations of the plasmids with point mutations were determined using a Nanodrop.

Nanodrop

BioBrick	Concentration (ng/µl)
SulA (pSB1C3)	167.7
BolA_ind (pSB1C3)	109.1
BolA_con (pSB4A5)	130.4
INP_Sil_Sdom (pSB4A5)	166.7

The PCR to correct the mutations was done according to the Phusion HF protocol. The elongation time was set at 75 seconds, and the annealing time was set at 57ºC. The PCR product was loaded on a 1% TAE agarose gel for electrophoresis, in order to determine of the PCR worked.

Left is a simulated image by SnapGene, right the actual gel picture. The lane marked with L is the Promega 1kb ladder, lane 1 contains SulA (pSB1C3), lane 2 contains BolA_ind (pSB1C3), lane 3 contains BolA_con (pSB4A5) and lane 4 contains INP_Sil_Sdom (pSB4A5)

The bands for BolA_ind, BolA_con and INP_Sil_Sdom are correct (lanes 2-4). The band for SulA (lane 1) is not as expected. This sample is checked on gel again.

electrophoresis

Left is a simulated image by SnapGene, right the actual gel picture. The lane marked with L is the Promega 1kb ladder, lane 1 contains SulA (pSB1C3. The other bands contain result from another experiment, irrelevant for this experiment.

The result from the second electrophoresis of SulA is still not as expected, we will start over with the correction of this BioBrick later. For now, we continue with BolA_ind (pSB1C3), BolA_con (pSB4A5) and INP_Sil_Sdom (pSB4A5).

BolA_ind (pSB1C3), BolA_con (pSB4A5) and INP_Sil_Sdom (pSB4A5) wetre purified according to protocol. The obtained concentrations were measured using Nanodrop.

Nanodrop

BioBrick	Concentration (ng/µl)
BolA_ind (pSB1C3)	99.9
BolA_con (pSB4A5)	98.5
INP_Sil_Sdom (pSB4A5)	61.5

The purified PCR product was restricted with DpnI, to restrict all methylated DNA containing the point mutation, leaving us with the corrected DNA. Restriction was done according to protocol. After 1 hour of incubation at 37ºC the restriction products were purified according to protocol. The prified restriction products were stored at -20ºC.

10^th August 2016

Tessa

Restricted PCR product of 3rd August, J23100 E0840, J23113 E0840 and J23117 E0840, with EcoRI-HF and PstI.

Purified restriction product.

Nanodropped purified product.

Nanodrop

Product	Concentration (ng/µl)
J23100 E0840	14.7
J23113 E0840	21.3
J23117 E0840	23.3

Ligated purified product into pSB1C3.

Transformed ligation product into TOP10 strain. Using RFP as positive control and sterile MilliQ as negative control. Plated on plates with LB agar and CM.

Lara

Of the previously sent DNA samples to sequencing, mVenus-pSB1C3, mKate-pSB1C3 and phaP-pSB1C3 were sequence confirmed. LacI-pSB1C3 and K1149051 were analysed again. Since the previous selected colonies for LacI-pSB1C3 turned out to contain the construct of the positive control RFP, a new colony-PCR was performed. Besides that, colony 47 of August 4^th was also transferred into liquid medium.

Via Phusion-PCR, the promoters J23100, J23105, J23108, J23113 and J23117 were added to the yesterday sequence confirmed phaP-pSB1C3. The melting temperature of the designed primers were for all 58 degrees Celsius; the template concentration was 102.0 ng/µl and the final construct should obtain 835 bp. The Phusion-PCR ran overnight.

Charlotte

The concentrations of the previously purified restriction products were measured using a Nanodrop:

Nanodrop

Product	Concentration (ng/µl)
BolA_ind (pSB1C3)	16.5
BolA_con (pSB4A5)	14.4
INP_Sil_Sdom (pSB4A5)	9.5

The restriction products were blunt-end ligated in order to circularize the plasmids. Since our ligation tool does not calculate the concentrations needed for blunt-end ligation, the following protocol was used:

Blunt-end ligation protocol

Component	Amount
T4 ligation buffer (10X)	2 µL
DNA	~80 ng
T4 Ligase	1 µL
Nuclease-free water	up to 20 µL

The T4 ligase was added last, the ligation mixture was left to incubate for 4 hours. After incubation, the ligation product was transformed into homemade chemically competent TOP10 cells, according to protocol. The tranformations were set to incubate overnight at 37ºC

11^th August 2016

Tessa

Colony PCR'd colonies from yesterdays transformation using Q5^&reg mastermix.

Ran a 1% agarose gel of the PCR product.

L is the Promega 1kb ladder, 1-6 is J23100 E0840, 7-14 is J23113 E0840 and 15-22 is J23117 E0840.

Transferred colony 14 (J23113 E0840 pSB1C3) and 21 (J23117 E0840 pSB1C3) into liquid LB.

Lara

Plasmids were isolated from the colonies that were grown overnight.

Nanodrop

DNA construct	Colony number	Concentration (ng/µl)
LacI-pSB1C3	47	56.5
LacI-pSB1C3	51	86.4
J23117 E0840	54	141.4

LacI-pSB1C3 was sent to sequencing.

Charlotte

Picked 8 colonies for the plates containing cells tranformed with BolA_ind (psB1C3) and 8 for BolA_con (pSB4A5). The colonies on the plate containing cells transformed with INP_Sil_Sdom were still too small to pick so they were left to incubate at 37ºC a little longer before picking 8 colonies for colony PCR.

The colony PCR was done according to protocol, using DreamTaq polymerase. The PCR products were loaded on a 1% agarose gel for electrophoresis. Colony 16 didn't fit in the PCR machine anymore, so this colony is also not loaded on the gel.

Left is a simulated image by SnapGene, right the actual gel picture. For BolA_ind, colonies 2,3 and 4 had a band at the correct height. For BolA_con, colonies 11, 12 and 13 had a band at the correct height. For INP_Sil_Sdom, colonies 17, 19 and 20 were correct. These colonies were transferred to selective liquid LB and grown overnight in a shaker at 37ºC and 250 rpm.

Charlotte & Liza

Tested 15 µm fluorescent polystyrene beads in the MicroTime 200 confocal microscope to see if the microscope is able to establish lasing in a bead. We used a 1 mW laser with an excitation wavelength of 532 nm. The bead was imaged at different intensities to see if we were able to see a shift to lasing at higher excitation energies.

12^th August 2016

Tessa

Miniprepped colony 14 and 21 of yesterdays overnight culture.

Nanodropped miniprep product.

Nanodrop

Product	Concentration (ng/µl)
J23113 E0840 in pSB1C3	140.6
J23117 E0840 in pSB1C3	325.6

Cryostocked colony 14 and 21.

Send miniprep product for sequencing. [Sequence Confirmed]

Lara

The results for the sequencing of LacI-pSB1C3 were checked: For both colonies 47 and 51 there was a 124 bp long exact same insert in the middle of the LacI-gene. In colony 54, there were two point mutations. The first one at location 2215, changing an Arg into Leu; the second mutation was on location 2625 changing a Glu into a stopcodon.

These two point mutations need to be fixed. Since normal mutation fixing by PCR only handles one mutation at a time, a new strategy was thought of: Two different linear fragments should be made, both fixing one mutation and able to join together with the other part via Gibson Assembly. The primers were designed and ordered.

The Phusion-PCR products of the different promoters that were obtained were transformed into TOP10 cells. For this, 2µl DNA was used per transformation, RFP-pSB1C3 as a positive control and MilliQ as a negative control. All transformations were plated onto LB-Cm plates and stored at 37 degrees Celsius.

Charlotte

The DNA of the colonies that were grown overnight was isolated using miniprep according to protocol. The isolated DNA was stored at -20ºC. The remaining culture was cryostocked according to protocol and stored at -80ºC.

Charlotte & Liza

Did a second attempt to get lasing in a bead with a confocal microscope.

13^th August 2016

Lara

A colony PCR was performed for the transformations of the different promoters attached to phaP-pSB1C3. The expected amplification length is approximately 1kb. Four colonies per plate were analyzed in the colony PCR.

This gel showed that only colony 35 (lane 15) did not contain the right PCR-product. The other colonies were transferred to liquid LB-Cm and cultivated overnight at 37 degrees Celsius.

14^th August 2016

Lara

The overnight grown cultures of the different promoters attached to phaP-pSB1C3 were plasmid isolated.

Nanodrop

DNA construct	Colony number	Concentration (ng/µl)
J23100 phaP pSB1C3	33	83.1
J23100 phaP pSB1C3	34	80.4
J23105 phaP pSB1C3	25	101.6
J23105 phaP pSB1C3	26	113.0
J23105 phaP pSB1C3	27	97.4
J23108 phaP pSB1C3	18	93.4
J23108 phaP pSB1C3	19	120.0
J23108 phaP pSB1C3	20	100.0
J23113 phaP pSB1C3	9	94.3
J23113 phaP pSB1C3	10	85.0
J23113 phaP pSB1C3	11	110.0
J23117 phaP pSB1C3	1	101.2
J23117 phaP pSB1C3	2	100.5
J23117 phaP pSB1C3	3	110.0

These plasmids were sent to sequencing.

13^th August 2016

Lara

A colony PCR was performed for the transformations of the different promoters attached to phaP-pSB1C3. The expected amplification length is approximately 1kb. Four colonies per plate were analyzed in the colony PCR.

This gel showed that only colony 35 (lane 15) did not contain the right PCR-product. The other colonies were transferred to liquid LB-Cm and cultivated overnight at 37 degrees Celsius.

15^th August 2016

Charlotte

The concentrations of the previously isolated DNA was checked on Nanodrop. The following concentrations were measured:

Nanodrop

BioBrick	Colony number	Concentration (ng/µl)
BolA_ind (pSB1C3)	2	63.0
	3	61.8
	4	68.3
BolA_con (pSB4A5)	11	101.9
	12	71.7
	14	90.9
INP_Sil_Sdom (pSB4A5)	17	88.0
	19	74.4
	20	43.2

The isolated DNA was prepared and sent for sequencing, according to protocol.

16^th August 2016

Charlotte

Sequencing had the following results:

BolA_ind (pSB1C3): All colonies had large inserts or deletions, so they couldn't be used for further experiments. We will try to repair the mutations again later.

BolA_con (pSB4A5): Colony 14 has an insert and colony 11 and 12 have a small gap. For colony 11 this is probably a readout error, when you look closer to the chromatogram. We can use colony 11 for further experiments.

INP_Sil_Sdom (pSB4A5): Colony 17 has a nonsilent mutation in the promoter and colony 20 has a gap in the promoter. Colony 19 has one silent mutation and one nonsilent mutation, but in the linker region. We can use colony 19 for further experiments.

17^th August 2016

Charlotte & Liza

Did a third attempt to get lasing in a bead with a confocal microscope.

18^th August 2016

Tessa

Transformation of the InterLab Study plasmids into BL21.

19^th August 2016

Tessa

Only Test Device 2 and 3 and positive control have yielded (very little) colonies. Decided to try and tranform them into Top10.

Lara

A colony PCR was performed for the transformation of LacI-pSB1C3.

Since none of the colonies yielded a positive result in the colony-PCR, the colony-PCR was performed again. This time with a new protocol, first boiling the colonies and using GoTaq buffer. A total of 5 µL DNA mixture was used.

22^nd August 2016

Tessa

Transformation of the InterLab Study plasmids into Top10.

Colony PCR of J23100 E0840 pSB1C3 using GoTaq^&reg. Plate from 11th August.

Ran a 1% agarose gel to verify part size

L is the Promega 1kb ladder, 1-9 are J23100 E0840.

Colony 1,2 and 3 were put in liquid LB+CM and incubated overnight.

Lara

A miniprep was performed to isolate plasmids from the LacI transformation, induced with IPTG. Concentrations can be found in the table below.

Nanodrop

DNA construct	Colony number	Concentration (ng/µl)
LacI pSB1C3	11	95.5
	K1149051 pSB4A5	2	120.3

The LacI pSB1C3 plasmid was sent to sequencing, the K1149051 pSB4A5 plasmid yielded a too low concentration during plasmid isolation, since the sequencing company requested higher concentrations for larger plasmids. The BioBrick K1149051 pSB4A5 was cultivated from a cryostock overnight and for the LacI pSB1C3 BioBrick precautional cryostocks were made.

23^rd August 2016

Tessa

Colony PCR of the InterLab Study plates using DreamTaq^&reg. Picked eight colonies of each plate.

Cryostocked J23100 E0840 pSB1C3 in Top10.

Ran a 1% agarose gel of the InterLab Study colony PCR products.

L is the Promega 1kb ladder, 1-8 is Test Device 1, 9-16 is Test Device 2, 17-24 is Test Device 3, 25-32 is Negative Control and 33-40 is Positive control.

Colony 1, 2, 4, 9-11, 17-19, 26, 27, 29 and 33-36 were picked for overnight culture in liquid LB+CM.

Lara

Plasmids were isolated for the yesterday overnight cultured K1149051 pSB4A5 and for different colonies of J23100 E0480 pSB1C3.

Nanodrop

BioBrick	Colony number	Concentration (ng/µl)
K1149051 pSB4A5	2	328.7
	J23100 E0480 pSB1C3	1	83.8
		2	89.5
3		85.4

These plasmids were sent to sequencing.

Primers that were needed for Gibson Assembly of the fluorophores and PHB constructs were diluted.

24^th August 2016

Tessa

Put InterLab Study cultures in fridge for later use.

Charlotte

Started a second round of correcting mutations in our designed BioBricks using PCR. The BioBricks that will be corrected are SulA (pSB1C3) BolA_ind (pSB1C3), INP_Sil_Sdom (pSB1C3) and OmpA_Sil_Sdom (pSB4A5). Prior to starting the correction of the mutations using PCR, the concentrations of these BIoBricks were measured on the Nanodrop:

Nanodrop

BioBrick	Concentration (ng/µl)
SulA (pSB1C3)	167.6
BolA_ind (pSB1C3)	109.1
INP_Sil_Sdom (pSB1C3)	139.2
OmpA_Sil_Sdom	170.8

The primers were diluted according to the manufacturers' instructions to a stock of 100mM. These stocks were consequently 10 times diluted to a working stock of 10mM.

Lara

Our advisor Esengül provided us with a protocol for the Gibson Assembly (see protocols).

The first step of the Gibson Assembly was to generate linear DNA segments by Phusion PCR. This was performed and to check the products, 5µL reaction product was loaded onto a 1% agarose gel.

25^th August 2016

Tessa

Resuspended FITC according to InterLab protocol.

Charlotte

Ran the PCR to fix the mutations in the BioBricks according to the Phusion HF PCR protocol, with an annealing time of 57ºC and and elongation time of 1.5 minutes. The results of the PCR were confirmed on gel, according to the electrophoresis protocol.

Left is a simulated image by SnapGene, right the actual gel picture. The lane marked with L is the Promega 1kb ladder, lane 1 contains SulA (pSB1C3), lane 2 contains BolA_ind (pSB1C3), lane 3 contains INP_Sil_Sdom (pSB1C3) and lane 4 OmpA_Sil_Sdom (pSB4A5)

The bands from BolA_ind (pSB1C3), INP_Sil_Sdom (pSB1C3) and OmpA_Sil_Sdom (pSB4A5) are at the correct height compared to the simulation. The lane containing SulA shows the same result as the previous attempt to correct this BoBrick. PRobably something goed wrong in the PCR, it's likely that the primers anneal at the worng position. We will not continue this BioBrick and use SulAin another backbone. The other PCR products were purified according to protocol and the concentrations were measured using Nanodrop.

Nanodrop

BioBrick	Concentration (ng/µl)
BolA_ind (pSB1C3)	107.7
INP_Sil_Sdom (pSB1C3)	88.7
OmpA_Sil_Sdom	100.3

The purified PCR products wererestricted with DpnI to remove any methylated and thus mutated DNA, according to the restriction protocol. Since our heat block was broken the samples were incubated in the stove at 37ºC. The restriction productswere purified according to protocol and stored at -20ºC.

Lara

Analysis of yesterday's gel: The products were not as expected. It appeared that the sequence of K1149051 was not what we expected as well. We figured that the backbone could have been pasted to eachother twice, forming a plasmid like that with antibiotic resistance. For this, another round of colony PCR for K1149051 pSB4A5.

26^th August 2016

Lara

The colony PCR of yesterday was gel analysed. The products in lanes 2, 3, 4, 9 and 10 were assumed to be correct. The corresponding colonies were transferred to LB+Amp and incubated overnight at 37 degrees.

28^th August 2016

Lara

Plasmid isolation was performed for the K1149051 pSB4A5 BioBrick.

Nanodrop

BioBrick	Concentration (ng/µl)
BolA_ind (pSB1C3)	14.7
INP_Sil_Sdom (pSB1C3)	11.9
OmpA_Sil_Sdom	12.6

29^th August 2016

Tessa

Transformed J23100-, J23105-, J23108-, J23113- and J23117 E0840 pSB1C3, and BolA_con pSB4A5 into BL21.

Charlotte

Checked the concentrations of the purified restriction products on Nanodrop:

Nanodrop

BioBrick	Concentration (ng/µl)
BolA_ind (pSB1C3)	14.7
INP_Sil_Sdom (pSB1C3)	11.9
OmpA_Sil_Sdom	12.6

The restriction products were blunt-end ligated according to the ligation protocol. The ligated BioBricks were transformed into homemade TOP10 cells, according to protocol. For the controls only 1/3 of the colume was used due to high transformation efficiency observed before. The transformants were plated on selective plated and left to incubate overnight at 37ºC.

30^th August 2016

Lycka

PCR lineralization of vectors and inserts for gibson assembly pha operon (BBa_K1149051) to fluorophores (GFP, mVenus, mKate, mCerulean). Phusion polymerase. Annealing temperature 60 degrees, elongation time 2 minutes.

Tessa

Did the InterLab Study plate reader measurements according to the InterLab plate reader protocol.

Streaked J23105- and J23117 E0840 pSB1C3 out on new plates, because they had too many colonies.

Colony PCR'd six colonies of BolA_con, J23100 E0840, J323108 E0840 and J23113 E0840 using GoTaq^&reg.

Ran a 1% agarose gel of PCR products to verify part size.

L is the Promega 1kb ladder, 1-6 is BolA_con, 7-12 is J23100 E0840, 12-18 is J23108 E0840 and 19-24 is J23113 E0840.

Picked colony 1, 6, 11, 13, 18, 20 and 21 for overnight culture.

Charlotte

Of each transformation plate, 8 colonies were picked: Colonies 1-8 were of BolA_ind, colonies 9-16 of OmpA_Sil_Sdom and colonies 17-24 of INP_Sil_Sdom. A colony PCR was performed with the picked colonies, according to the GoTaq Colony PCR protocol. The annealing time was 1.5 minutes.

The PCR products were checked on gel according to the Electrophoresis protocol, which yielded the following gel:

L is the Promega 1kb ladder, 1-8 is BolA_ind, 9-16 is OmpA_Sil_Sdom, 17-24 is INP_Sil_Sdom

Colonies 1, 3, 4, 14, 15, 16, 20, 21 and 22 were picked and transferred to selective medium for an overnight culture, according to protocol.

31^st August 2016

Lycka

Electrophoresis of PCR fragments yesterday.

Picture on the left shows simulated agarose gel. Picture on the right shows the actual agarose gel. 1 - 4: vectors for mCerulean, mKate, mVenus and GFP, respectively. 5 - 6 inserts for mVenus and GFP. Gel did not yield bands at the right height. PCR is repeated at an annealing temperature of 57 degrees.

Second gel also did not yield the right bands. PCR repeated overnights by María at Tm 64 degrees and elongation time 3.5 minutes in the presence of DMSO.

Transfer from cryostock to plate BL21 strains with fluorophores expressed. On LB agar supplemented with chloramphenicol.

Tessa

Cryostocked J23100-, J23108- and J23117 E0840 pSB1C3 in BL21. BolA_con didn't grow.

Cryostocked K1149051 pSB4A5 in BL21.

Charlotte

The overnight cultures of BolA_ind (pSB1C3), OmpA_Sil_Sdom (pSB4A5) and INP_Sil_Sdom (pSB1C3) were cryostocked according to protocol, 3 tubes per culture. The remaining culture was used for DNA isolation by Miniprep, according to protocol. Two tubes per colony were prepared. The concentration of the isolated DNA was measured using Nanodrop, the following concentrations were measured:

Nanodrop

BioBrick	Colony number	Tube 1 concentration (ng/µl)	Tube 2 concentration (ng/µl)
BolA_ind (pSB1C3)	1	91.2	113.5
	3	87.9	79.6
	4	104.5	87.7
OmpA_Sil_Sdom (pSB4A5)	14	133.5	143.7
	15	152.5	141.4
	16	171.1	159.4
INP_Sil_Sdom (pSB4A5)	20	109.6	90.6
	21	112.2	101.5
	22	112.0	111.9

The isolated DNA was prepared and sent for sequencing, according to protocol.

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September

1^st September 2016

María

PCR with new primers of those parts that need to be expressed under the lac promoter. These primers will allow cloning on Addgene plasmids from the pBb series. The plasmids chosen (pBbS5a and pBbA5c) express lacI and the cloning site is placed downstream of a lac promoter. The parts to be amplified are:
1. BolA
2. INP_Sil_Sdom
3. OmpA_Sil_Sdom
4. OmpA_Sill_Taur
5. Sil_Sdom
6. SulA

Lycka

Electrophoresis of overnight PCR for lineralization.

Picture on the left shows simulated agarose gel. Picture on the right shows the actual agarose gel. 1 - 4: vectors for mCerulean, mKate, mVenus and GFP, respectively. 5 - 6 inserts for mVenus and GFP. For all lanes except lane 5, the right bands are present. Also many other bands were present so the product was gel purified.

Transfered one colony of BL21 on each plate from yesterday in a 50mL falcon tube with 10mL filter sterilized M9 medium with glucose supplemented with chloramphenicol.

Tessa

Put three colonies of J23100-, J23105- and J23117 E0840 pSB1C3 in BL21 into liquid LB+CM.

2^nd September 2016

Tessa

Cryostocked J23100-, J23105- and J23117 E0840 pSB1C3 in BL21.

Lycka

Nanodrop gel purified PCR fragments from yesterday. Concentrations are too low to work with. Repeat PCR under the same conditions, but with double volume (100µL). Done by María.

Measure OD600 of cultivated M9 tubes.

OD600 measurements

Genotype	OD600
GFP J23108	1.24
mVenus	0.04
mKate	0.20
mCerulean	0.03

The strains containing GFP and mKate grew really well, the ones containing mVenus and mCerulean did not. All of the tubes were put back in the incubator. The one with GFP was diluted 10 times in M9 medium, the one with mKate was diluted 2 times. Aliquots of 150µL were loeaded in an 96 well plate and measured in a plate reader at 37 degrees for 7 hours.

María

PCR of same samples as Lycka in a reaction volume of 100µL. After gel isolation and purification only for mCerulean and mKate good concentrations were reached (160 and 260 ng/µL, respectively), for the rest of samples either no bands were seen in the agarose gel or the concentration was too low again.

Charlotte

Analyzed sequencing results. Both the colonies of BolA_ind and OmpA_Sil_Sdom had an insert around the mutation spot, so sequences were not correct. Also all colinies of INP_Sil_Sdom had non-silent mutations.

3^rd September 2016

Lycka

The cultures in M9 grew well in the incubator until an OD of approximately 1. Samples were diluted 10 times in M9 supplemented with chloramphenicol and measured in the plate reader.

6^th September 2016

Lycka & Lara

Colony PCR for the transformants of the Gibson Assembly.

Most colonies appear to be false positives, since the bands correspond to the length of the vector only. Colonies 43 and 44 might contain the desired plasmid (with the PHB operon fused to mKate), so these are transfered to LB medium.

8^th September 2016

Lycka

Miniprep, cryostock and prepare for sequencing colonies 43 and 44. The concentrations were 252.6 and 242.6 ng/µl, respectively.

9^th September 2016

Lycka

In order for the cells to grow better, we prepared enriched M9 medium (eM9), instead of regular M9. The medium was supplemented with chloramphenicol and devided into aliquots of 10 mL. From all plates with fluorophore expressing cells, one colony was picked and grown in eM9 overnight.

10^th September 2016

Lycka

Transfer to LB cells with plasmids bought from Addgene: pBbS5a and pBbA5c

Measure OD of overnight cultures in eM9. Dilute them until OD of 0.1 in eM9 supplemented with chloramphenicol. Measure kinetic cycle of 7 hours in plate reader.

11^th September 2016

Charlotte & Lara

Miniprepped plasmids bought from Addgene pBbS5a and pBbA5c from overnight LB cultures; 5 tubes each. The concentrations were confirmed with nanodrop and were between 30 and 60 ng/µL.

María

Restriction of plasmids bought from Addgene pBbS5a and pBbA5c, and PCR products from September 1st with EcoRI and XhoI. The biggest fragment obtained with each plasmid is the backbone, which was gel isolated. Then, backbones and fragments were ligated and transformed into Top10 chemically competent cells.

Tessa

Transformation of

BolA_con pSB1C3
INP_Sil_Sdom pSB4A5
OmpA_Sil_Taur pSB4A5
Sil_Sdom pSB4A5
Sul_A pSB1C3
OmpA_Sil_Sdom pSB4A5
RFP pSB1C3 (positive control)
RFP pSB4A5 (positive control)
Sterile MiliQ (negative control)

in TOP10. Plated on selective agar plates.

12^th September 2016

María

Colony PCR of transformations from yesterday 16 colonies of each transformation were picked, but only analized 15 of each.

Red arrows point at colonies picked for overnight cultures in selective medium.

Lycka

Measure spectrum of mVenus and mCerulean cells in plate reader.

13^th September 2016

María

Plasmid isolation, preparation of samples for sequencing and cryostock of overnight cultures of colonies picked yesterday.

Sequences of all colonies picked for OmpA_Sil_Taur, INP_Sil_Sdom, Sil_Sdom and SulA were confirmed to be good. In the case of BolA there's a deletion of a single bp, to be fixed by PCR. All sequences for OmpA_Sil_Sdom contained a strange long insert, this silicatein gene version was dropped for further experiments.

Tessa

Put 100µl of TOP10 competent cells into 10ml LB in a falcon tube and incubated overnight at 37 degrees Celsius at 250rpm.

Made LB and LB agar.

Lycka

Measure spectrum of eM9 cultures in plate reader.

Sequences of mKate gibson assembled to phaCAB arrived and didn't yield the good results.

14^th September 2016

Tessa

Made ~100 aliquots TOP10 competent cells with culture from yesterday according to protocol. OD600 before putting on ice was 0.34.

15^th September 2016

María

Transformation of plasmids with confirmed sequences from 13^th of September into BL21. Also, combined with GFP under promoter J23100 in pSB1C3 backbone. All 3 silicatein versions, SulA and the combination of Sil_Sdom and GFP yielded colonies. Other 2 silicateins together with GFP did not yield any positive result.

Tessa

Did positive and negative controls of competent cells made yesterday. Used RFP pSB1C3 for positive control and MiliQ for negative control.

Lycka

Wash 3x in PBS cultures grown in LB expressing fluorophores. Dissolve in PBS and measure spectrum in plate reader.

16^th September 2016

Lycka

Repeat kinetic cycle experiment in plate reader with different settings, because the previous results didn't show good results.

18^th September 2016

Lycka

Transfer from plate to liquid LB colonies with BL21 cells expressing OmpA_Sil_Taur, Sil_Sdom and INP_Sil_Sdom for widefield microscope experiment.

19^th September 2016

Lycka & María

Transfer LB cultures from yesterday to fresh medium and grow until OD reached 0.65. Then added 1mM sterile IPTG to induce. Grown in the incubator for another 5 hours. Subsequently added 60µM Sodium Silicate and incubated for 3 hours before putting them in the fridge.

Diluted and filter sterilized Sodium Silicate in miliQ water up to a concentration of 1M.

Diluted and filter sterilized Rhodamine 123 in ethanol up to a concentration of 20 mg/mL and added 15µL of this to the indicated cultures.

20^th September 2016

María

Transfer of colonies from 15^th of September transformations into overnight culture for cryostocking.

Lycka & Charlotte

Add 7µM rhodamine to the cultures from yesterday, incubated 10 min. Centrifuged 1.5 mL of culture at 4000 rpm 5 min Washed 3x and then resuspended in 400µL PBS. Imaged in widefield microscope with excitation wavelength of 488 nm.

21^st September 2016

María

Cryostock of BL21 strains containing sequence confirmed plasmids from yesterday's overnight cultures.

Transformation into BL21 of combinations of OmpA_Sil_Taur with all fluorophores (separately) and INP_Sil_Sdom with GFP under promoter J23100.
None of them yielded any colonies. We are going to try reducing the antibiotics concentration in the plates to half because probably the cells are not able to handle the 2 plasmids and such a high concentration of antibiotics.

Lycka

Measure OD600 of overnight cultures María prepared. They were around 1.46, 1.35, 1.49 for Sil_Sdom, OmpA_Sil_Taur, INP_Sil_Sdom respectively. 1mL of culture was transfered to 10 mL fresh LB.

Dissolved and filter sterilized IPTG. 1.2 g in 5 mL.

Saved cultures in the fridge to continue tomorrow.

22^th September 2016

Lycka

Inoculate 0.25 mL of yesterday's culture in 10 mL fresh LB. Cells did not grow after half a day. Inoculate new culture from plate, grown overnight.

23^th September 2016

Lycka

Test silicic acid test, with 1mM aliquot of silic acid.

100 µL 1 mM silicate
4900 µL miliQ
3 drops reagent 1
3 drops reagent 2
500 µL reagent 3

24^th September 2016

Lycka

Measure OD600 of overnight eM9 cultures, diluted 4 times before measuring.

OD600 measurements

Genotype	OD600
GFP J23100	0.90
GFP J23105	0.51
GFP J23108	0.88
GFP J23113	didnt grow
GFP J23117	didnt grow
mVenus	0.74
mCerulean	0.77

Dilute until OD 0.1 and divide into aliquots of 100µL in a 96 well plate. Performed kinetic cycle in plate reader.

26^th September 2016

María

Repeat of transformations of 21^st of September using plates with half the concentration of antibiotics. All of them yielded colonies.

27^th September 2016

María

Transfer of colonies from yesterday's transformations to overnight culture.

Lycka

Prepare eM9 an divide into aliquots of 10 mL, inoculate with:

OmpA Sil Taur
INP Sil Sdom
Sil Sdom
BolA
OmpA Sil Taur
OmpA Sil Taur + mCerulean
mCerulean

Restreak plates with BL21 OmpA Sil Taur and BolA on fresh LB+CM plates.

Cryostock OmpA + mCerulean, + mVenus and + all GFPs.

28^th September 2016

María

Cryostock of BL21 cells from overnight culture from yesterday.

Fixing of BolA mutation by PCR.

Picture on the left shows simulated agarose gel. Picture on the right shows the actual agarose gel. PCR didn't work, we are going to try again with a gradient of annealing temperatures to see if this is the cause of the problem.

Lycka

Measure OD600 of overnight eM9 cultures, diluted 4 times before measuring.

OD600 measurements

Genotype	OD600
INP Sil Sdom	1.15
BolA	1.18
OmpA Sil Taur	1.26
Sil Sdom	1.00

Dilute until OD 0.05 in 200 mL eM9 and grown in shaker at 37 degrees. Cultures didn't grow after half a day.

Prepared calibration curve for silicate test. Dilutions made with 1 mM silic acid stock.

Calibration curve

concentration (µM)	silica stock (µL)	miliQ (µL)
100	500	4500
80	400	4600
60	300	4700
40	200	4800
20	100	4900
10	50	4950
5	25	4975
0	0	5000

Prepare overnight eM9 cultures for:

Silic acid test: OmpA_Sil_Taur, INP_Sil_Sdom, Sil_Sdom, BolA
Laser setup: mCerulean, OmpA_Sil_Taur, mCerulean + OmpA_Sil_Taur
TEM: OmpA_Sil_Taur, INP_Sil_Sdom, Sil_Sdom

29^th September 2016

Tessa

Made ~50 aliquots BL21 competent cells according to protocol. OD600 before putting on ice was 0.30.

María

PCR to fix BolA mutation.

All annealing temperatures resulted in a product of the desired length.
1. 66ºC; 2. 65ºC; 3. 63.4ºC; 4. 60ºC

Lycka & María

Measure OD600 of overnight eM9 cultures, diluted 4 times before measuring.

OD600 measurements

Genotype	OD600
INP Sil Sdom	0.77
BolA	0.74
OmpA Sil Taur	0.75
Sil Sdom	0.68

Inoculate 4 µL in 150 µL fresh eM9. After induction Erlenmeyer fell over and content was not usefull anymore.

Measure OD600 of overnight LB cultures, diluted 4 times before measuring.

OD600 measurements

Genotype	OD600
INP Sil Sdom	0.73
OmpA Sil Taur	0.76
Sil Sdom	0.91

Inoculate 100 µL in 10 mL fresh medium. Grow until OD 0.55 and add 10 µL 1 M IPTG.

30^th September 2016

María

Silicic acid test: since cells grew too slow in eM9 medium we checked if LB absorbance spectrum interfered with the absorbance of the final product of the test (~815nm). It didn't so we decided to grow the cells in LB medium.
Measured OD600 of overnight cultures with a 1/4 dilution:

Genotype	OD600
OmpA Sil Taur	0.77
INP Sil Sdom	0.74
Sil Sdom	0.75
BolA	0.68

Carmen & Charlotte

Tested whether adding silicatein cells to a solar panel had any effect. We tested the strain expressing OmpA-fused silicatein which was incubated with silica, so it had a silicate coating. The cells were washed 3x in PBS. We connected a multimeter that was measuring voltage to a solar cell with a limited area exposed to light. We measured the output power of the solar cell at the same time as the light intensity of the lamp. We repeated this experiment with the same area of the solar cell covered in PBS and covered in PBS + silicatein-expressing cells.

Charlotte & Liza

Tested the mCerulean-expressing cells in our self-built hardware setup. The cells were grown in LB and washed 3x with PBS. The cells were fixed on a slide by pipetting them under 3% agarose-TAE pads.

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October

4^th October 2016

Charlotte & Liza

Tested the mCerulean-expressing cells, the OmpA-silicatein expressing cells and OmpA-silicatein + mCerulean expressing cells in our self-built hardware setup. The cells were grown in LB, spun down and resuspended in PBS. The cells were fixed on a slide by pipetting them under 3% agarose-TAE pads. The cells were excited at 405 nm and imaged at different excitation energies.

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Protocols

During our lab work we used the following protocols.

Chemically competent cells

Grow overnight culture from single colony or competent cells aliquot.
Inoculate 1:100 in the desired volume of LB.
Grow to OD 600 ~0.35 (important that it is close to 0.35, 0.3-0.4 is good).
Chill on ice for 20 min (it can chill for a 1-2 hours).
Centrifuge at 8000g for 8 minutes in Falcon tubes, decant supernatant.
Resuspend by pipetting gently in 1/10 (of the volume of LB from step 2) of ice-cold TSS.
NOTE: It can take a while to break up the pellet (you can use a 25 mL pipette and scrape the pellet off the side of the tube before pipetting).

10 g PEG 3350
5 mL DMSO
2 mL 1 M MgCl₂
LB to 100 mL

Freeze 100 µL aliquots in liquid nitrogen & store at -80 degrees C in 1,5mL Eppendorf tubes.
ALWAYS do a transformation positive control using a plasmid that you know is efficiently taken up by competent cells to confirm competency.
Just as important: ALWAYS do a negative control transformation (add untransformed cells to a plate with antibiotics) to confirm that you don't have an antibiotic-resistant contaminant strain in your competent cell stock. This happens from time to time and can cause lots of agony!

PCR (Phusion polymerase)

Prepare the mix for all the samples in a single 1.5mL tube (mind pipetting error!). For one sample:

Component	Volume (µL) for 20µL reaction	Volume (µL) for 50µL reaction
5X Phusion HF Buffer	4	10
10mM dNTPs	0.4	1
FW Primer	1	2.5
RV Primer	1	2.5
Phusion polymerase	0.2	0.5
Template DNA (<250ng)	Variable	Variable
Nuclease-free H₂O	Up to 20µL	Up to 50µL

NOTE: The 250ng of template DNA are approximate, choose a volume that works fine for all your samples and fill it in the excel sheet so you are able to prepare a mix for all samples at the same time.

Add the total volume per sample (without DNA) to each PCR tube.
Add the template DNA to each tube.

Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):

Step	Temperature (ºC)	Time
Initial denaturation	98	30sec
Denaturation	98	10sec
Annealing	Tm-3	20sec	x30 cycles
Extension	72	15-30sec/1kb	x30 cycles
Final extension	72	5min
	4	Hold

Run a DNA electrophoresis following the DNA electrophoresis protocol.

Gel isolation

Run a DNA electrophoresis (following the DNA electrophoresis protocol), loading the full volume of PCR or restriction product into the agarose gel. If necessary, use multiple wells.
Cut the band (or bands) corresponding to the fragment of interest with a razor blade and put it in a pre-weighted 1.5mL tube, then calculate the weight of the gel fragment.
Add 10µL of Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50-65ºC until gel slice is completely dissolved.
Pre-warm an aliquot of Nuclease-free water at 50-65ºC.
Insert SV Minicolumn into Collection Tube and label both of them according to the labelling of your samples.
Transfer the dissolved gel mixture to the Minicolumn assembly. Incubate at room temperature for 1 minute.
NOTE: When pipetting into the column, aim the pipette to the wall not the membrane to avoid damaging it.
Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.
Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.
Add 700µL of Membrane Wash Solution (if it is the first use, dilute it with 95% ethanol following the bottle’s instructions).
Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.
Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.
Repeat steps 9-11 with 500µL of Membrane Wash Solution and centrifuging for 5 minutes.
Once the Collection Tube is empty, centrifuge the Minicolumn assembly at maximum speed for 1 minute with the microcentrifuge lid open to allow ethanol full evaporation.
Transfer the SV Minicolumn to an empty 1.5mL tube carefully labelled.
Add 500µL of the pre-warmed water (300µL for higher concentrations or when small amounts of DNA are suspected) directly to the centre of the SV Minicolumn, without touching the membrane with the pipette tip.
Incubate at room temperature for 5 minutes.
Centrifuge at maximum speed for 1 minute.
Discard the SV Minicolumn, cap the tube containing the eluted DNA and keep at 4ºC (for immediate use) or -20ºC (for storage).

Restriction

Get the detailed protocol for restriction from the following website, according to the enzymes used. When high restriction product concentrations are needed (generally for backbones) the amount of DNA can be increased to 3-5g. The volume of DNA necessary can be obtained from the concentration measured at NANODROP.
Mix all the components stated by this restriction tool in a 1.5mL Eppendorf tube and incubate at 37ºC for 1-2h.

Ligation

Make your own ligation protocol.

Mix all the components in a tube (always add the T4 ligase at the end).
Incubate for at least 3 hours at 16ºC.

Transformation (Top10 chemically competent cells)

Get as many aliquots of competent cells (100L) from the -80ºC freezer as transformations to be done and put them on ice for 10-15min.
NOTE: Don’t forget positive (RFP in the backbone you are using, check plasmids box in the -20ºC freezer) and negative controls (no DNA).
Add to the 100µL of competent cells:
1. 1µL of plasmid or 2µL of ligation product.
2. 20µL 5X KCM buffer.
3. Sterile water up to 200µL.
Mix by flicking the tube.
Incubate on ice for 20-30 minutes.
Heat shock at 42ºC for exactly 90 seconds.
Return the cells to ice for 1-2 minutes.
Add 400µL of fresh LB.
Incubate at 37ºC with shaking for 1 hour.
NOTE: Not at the thermoblock, use a shaking incubator.
To increase the concentration of cells before plating, centrifuge the tubes at 4000rpm for 3 minutes, remove 500L of medium, resuspend the pellet in the remaining volume.
Plate the rest of the volume of the transformation on selective LB plates and incubate at 37ºC overnight.

Transformation (BL21 competent cells)

Put a tube of BL21 Competent E. coli cells on ice for 10 minutes.
Add 2µL of plasmid DNA to the cell mixture.
Mix by flicking the tube.
Place the tube on ice for 30 minutes.
Heat shock at 42ºC for exactly 10 seconds.
Place on ice for 5 minutes.
Add 950µL of SOC medium at room temperature.
Incubate at 37ºC with shaking for 1 hour.
NOTE: Not at the thermoblock, use a shacking incubator.
To increase the concentration of cells before plating, centrifuge the tubes at 4000rpm for 3 minutes, remove 700µL of medium, resuspend the pellet in the remaining volume.
Plate 70µL of the transformation on selective LB plates and incubate at 37ºC overnight.

Transformation (OneShot^® Top10 Chemically competent cells)

Get half the amount of tubes of OneShot^® TOP10 chemically competent cells (tubes with a purple lid) than the transformations to be done from the -80ºC freezer and put them on ice for 10-15min.
Divide the volume of competent cells of each tube in 2. Thus, transfer 25L to a new sterile tube.
Add 1µL of plasmid or 2µL of ligation product into each tube and mix by flicking.
Incubate on ice for 30 min.
Heat shock the cells for 30 seconds at 42ºC.
Put back on ice for 2 minutes.
Add 250µL of LB medium.
Incubate at 37ºC with shaking for 1 hour.
NOTE: Not at the thermoblock, use a shacking incubator.
To increase the concentration of cells before plating, centrifuge the tubes at 4000rpm for 3 minutes, remove 200µL of medium, resuspend the pellet in the remaining volume.
Plate the rest of the volume of the transformation on selective LB plates and incubate at 37ºC overnight.

Colony PCR (Q5^® High-Fidelity 2X Master Mix)

Choose the colonies to be picked for testing and number them. When available pick at least 8 colonies per plate, or more if there are slots left in the PCR machine (it fits 25 samples).
NOTE: It is more convenient if the numbering is continued between plates of the same batch instead of starting at 1 in each plate. This way there is only one set of numbers per date.
NOTE: it is possible that not all samples fit in one PCR and then more than one would need to be performed, so keep that in mind for the timing of experiments. But don’t be sad, that would mean that the transformation worked so well that you have a lot of colonies to choose from!! ☺

Prepare the mix for all the samples in a single 1.5mL tube (mind pipetting error!). For one sample:

Component	Volume (µL)
Q5^® HF 2X Master Mix	12.5
Primer VF2	1.25
Primer VR	1.25
Nuclease-free H₂O	10
Total	25

Pipette 25µL of mix into each PCR tube.
Under sterile conditions, pick a colony making sure that part of it stays on the plate for later use and soak it in the content of the PCR tube. When finished, put them back in the incubator so they grow for a bit longer while the PCR runs.

Put the tubes in the PCR machine and apply the following program (applicable for the primers mentioned above, for other sets of primers the annealing temperature needs to be adjusted):

Step	Temperature (ºC)	Time
Cell lysis and initial denaturation	95	15min
Denaturation	95	30sec
Annealing	53	30sec	x30 cycles
Extension	72	30sec/1kb	x30 cycles
Final extension	72	5min
	4	Hold

While the PCR is running, prepare a 0.5mL tube with 2µL loading buffer for each PCR sample and one tube with 4µL of 1Kb DNA ladder (or 8µL if 2 gels are needed). This way you can take these tubes into the EtBr area and don’t worry about how to handle the PCR tubes.
Cast an agarose gel following the DNA electrophoresis protocol.
Prepare an agarose gel with SNAPGENE for comparison with the actual results.
Once the PCR program is finished, transfer 8L of PCR product into the tubes containing the loading buffer and follow the DNA electrophoresis protocol.

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DNA electrophoresis

Work clean! Handle with gloves all material labelled as EtBr contaminated, don’t take it outside of the EtBr area and don’t touch with gloves anything that is not labelled as EtBr contaminated.

Prepare TAE buffer: take the 10X concentrated TAE from the chemicals cabinet and dilute it 10 times with demi water. For 500mL, add 50mL to 450mL of dH2O.
Weigh agarose for a 1% gel. For 200mL, 2g of agarose is necessary.
Mix the TAE solution with the agarose and heat the solution until it is completely dissolved.
Add Ethidium bromide to the gel mould. 2.5L for the small gel and 5L for the big one.
Pour the solution into the mould making sure there are no bubbles and that the EtBr is completely mixed. Let it solidify (approx. 20 minutes).
Transfer the gel to the electrophoresis cell minding the arrow that indicates the direction of DNA migration, remove the combs and cover it with TAE.
Prepare tubes with 5/6 of PCR product and 1/6 of loading buffer.
Load the molecular weight marker (ladder) in the first well (check the appropriate volume for each marker, generally 5L works fine) and load the samples in the other wells, according to the order in your lab journal.
NOTE: Do not contaminate the loading buffer and ladder with EtBr! Do not touch it while wearing a glove.
Connect the cables following the colour code and run at 100-130V for 30-60 min.

Restriction product purification

For restriction products containing more than one large fragment follow the first 3 steps of the Gel isolation protocol.
When restriction yields only one large fragment, add an equal volume of Membrane Binding Solution to the volume of restriction product.
Pre-warm an aliquot of Nuclease-free water at 50-65ºC.
Insert SV Minicolumn into Collection Tube and label both of them according to the labelling of your samples.
Transfer the dissolved gel mixture to the Minicolumn assembly. Incubate at room temperature for 1 minute.
NOTE: When pipetting into the column, aim the pipette to the wall not the membrane to avoid damaging it.
Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.
Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.
Add 700µL of Membrane Wash Solution (if it is the first use, dilute it with 95% ethanol following the bottle’s instructions).
Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.
Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.
Repeat steps 9-11 with 500µL of Membrane Wash Solution and centrifuging for 5 minutes.
Once the Collection Tube is empty, centrifuge the Minicolumn assembly at maximum speed for 1 minute with the microcentrifuge lid open to allow ethanol full evaporation.
Transfer the SV Minicolumn to an empty 1.5mL tube carefully labelled.
Add 500µL of the pre-warmed water (300µL for higher concentrations or when small amounts of DNA are suspected) directly to the centre of the SV Minicolumn, without touching the membrane with the pipette tip.
Incubate at room temperature for 5 minutes.
Centrifuge at maximum speed for 1 minute.
Discard the SV Minicolumn, cap the tube containing the eluted DNA and keep at 4ºC (for immediate use) or -20ºC (for storage).

Cell frozen stock

Work sterile! If plasmid DNA needs to be purified from the overnight culture, remove the necessary volume from the Falcon tube before the cryostock procedure.

Centrifuge the 50mL tubes at 2000rpm for 10 min.
Decant the supernatant without disturbing the pellet.
Add fresh LB medium, around 1/3 of the starting volume of the culture.
Mix by vortexing until the pellet is completely resuspended.
Add sterile 80% glycerol solution, the same volume as fresh LB in step 4.
Mix by vortexing.
Make 1.5mL aliquots in cryotubes and label them with the cell type, the BioBrick and backbone they contain (if applicable) and the date.
Store them at -80ºC and update the inventory in the drive folder.

Plasmid isolation

Pre-warm an aliquot of Nuclease-free water at 50-60ºC.
Add 1.5 mL of bacterial culture in LB medium to a 1.5 mL micro-centrifuge tube. Centrifuge that tube at max speed for 3 min.
NOTE: For higher concentrations repeat step 1 in the same tube.
NOTE: If cryostocks need to be prepared from the same overnight culture tube, remove the necessary volume for plasmid isolation under sterile conditions!
Remove the supernatant.
Resuspend the pellet in 600 µL of MiliQ water.
Add 100 µL of Cell Lysis Buffer, and mix by inverting 6 times. The color change to blue indicates complete lysis.
Add 350 µL of cold (4-8ºC) Neutralization Buffer, and mix by inverting the tube. The color change to yellow indicates total neutralization.
Introduce a PureYield Minicolumn into a PureYield Collection tube.
Centrifuge at maximum speed for 3 minutes, and transfer the supernatant to the Minicolumn assembly.
NOTE: When pipetting into the column, aim the pipette to the wall not the membrane to avoid damaging it.
Centrifuge at maximum speed for 15 seconds and discard the flowthrough.
Add 200 µL of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed for 15 seconds. It is not necessary to empty the Collection Tube now.
Add 400 µL of Column Wash Solution to the minicolumn, and centrifuge at maximum speed for 30 seconds.
Transfer the PureYield Minicolumn to a clean 1.5 mL tube, add 30 µL (or 50L if you expect high concentrations) of pre-warmed MiliQ water directly to the minicolumn matrix. Let stand for 5 minutes at room temperature.
Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute plasmidic DNA. Cap the tube, and store the DNA solution at -20 ºC (or keep it at 4ºC if it is to be used immediately).
NOTE: If possible, put the lids in line with the circle of the centrifuge (so not pointing out or inwards) so that the caps don’t break while centrifuging.

Overnight cell cultures

Prepare 20mL of selective LB (with the antibiotic your cells are resistant to) per colony to be picked.
NOTE: You can use a sterile bottle to prepare the total volume.
Transfer 10mL of selective LB to a 50mL Falcon tube. Prepare 2 tubes per colony: 1 for miniprep and 1 for cryostocks.
Pick 1 colony using an inoculation loop and soak it in the LB. Make sure you have cells left for the second tube.
Take the tubes to room F1.690 and incubate overnight at 37ºC in the shaking incubator.

Polysilicate layer in silicatein expressing cells

Inoculate a fresh culture of cells from an overnight culture. Make a dilution of around 1/100 of the overnight culture into fresh selective LB.
Incubate in the shaking incubator at 37ºC and 250 rpm until it reaches exponential phase (just measure OD600 from time to time until duplication time is close to 20 min).
Induce the culture with 1/1000 volume of IPTG 1M.
Incubate in the shaking incubator at 37ºC and 250 rpm for 3h.
Add sodium silicate to a final concentration of 60µM from the 1mM stock (maybe new stock needs to be made from the 1M stock, keep sterile!!).
Incubate at 37ºC and 250 rpm for 3-4h, after this the cells are ready for further experiments.

Growth study

Follow the Polysilicate layer protocol until step 5.
Take a 100µL sample directly after adding the sodium silicate, this is t=0.
Incubate at 37ºC and 250 rpm and take a 100µL sample every hour.
For each sample:
1. Make a series of dilutions in 1/10 steps until reaching a 1/10⁶. It can be done in a 96 well plate by adding 10µL of the previous dilution in 90µL of LB medium.
2. Plate in agar plates 20µL of dilutions 1/100, 1/10³, 1/10⁴, 1/10⁵, 1/10⁶.
Incubate at 37ºC overnight.
Hopefully, in some of the dilutions the colonies will be countable. Count colonies in plates for at least one dilution of each strain and time point to make a growth/viability curve.

Plate reader

Silicic acid test

For a 1 mL sample:

Add 20µL of reagent Si-1 and mix.
Wait for 3 min.
Add 20µL of reagent Si-2 and 100µL of Si-3 and mix.
Wait for 10 min.
Measure absorbance at 824nm.

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-                    <h2 class="title-style-1">Day Notes<span class="title-under"></span></h2>
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-                                    <h4 class="panel-title">
-                                        June
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-                            </a>
-                            <div id="collapseOne" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingOne">
-                                <div class="panel-body">
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">29<sup>th</sup> June 2016 </h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Prepare 10mL of antibiotic stocks of both antibiotics that are going to be used during the project: Chloramphenicol (Cm) and Ampicillin (Amp). Make 500µL and 1mL aliquots.</p>
-                                            <p>Prepare 1L of LB medium and 3 400mL bottles of LB Agar. Send to autoclave.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
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-                                        <h2 class="title-style-3">30<sup>th</sup> June 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Add antibiotics to the LB Agar bottles prepared the previous day and pour plates to have them ready for when we start transforming.</p>
-                                        </div>
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-                                    <!-- end activity-->
-                                    <a href="#notes" class="btn btn-info" role="button" onclick="$('html,body').animate({scrollTop: $('#notes').offset().top}, 'slow');" style="text-decoration:none; color:#f3f4f4; float:right;">Back to Top</a>
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-                            </div>
-                        </div>
-                        <!-- next month-->
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-                                    <h4 class="panel-title">
-                                        July
-                                    </h4>
-                                </div>
-                            </a>
-                            <div id="collapseTwo" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingTwo">
-                                <div class="panel-body">
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-                                    <div class="row">
-                                        <h2 class="title-style-3">4<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Digestion gBlocks mVenus and mKate with EcoRI and PstI.</p>
-                                            <p>Made liquid culture of strain containing pSB4A5 bakcbone with RFP.</p>
-                                            <p><strong>María</strong></p>
-                                            <p>Digestion of pSB1C3 linearized backbone from the distribution kit with EcoRI and PstI.</p>
-                                            <p><strong>Charlotte</strong></p>
-                                            <p>Made two liquid overnight cultures of <i>E. coli</i> K12 harboring the standard backbone pSB4A5 containing RFP.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
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-                                        <h2 class="title-style-3">5<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Dissolution of necessary parts from distribution kit: GFP BioBrick BBa_E0840 and promoters BBa_J23100, BBa_J23105, BBa_J23108, BBa_J23113 and BBa_J23117.</p>
-                                            <p>Dissolution of K1149051 (a kind gift of Imperial College), which was sent dry on filter paper.</p>
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Ligation of mKate into backbone pSB1C3.</p>
-                                            <p>Transformation of ligation product mKate and the BioBricks dissolved by María into <i>Escherichia coli</i> Top10 cells. These were plated on LB medium supplemented with Cm.</p>
-                                            <p><strong>Charlotte</strong></p>
-                                            <p>Did 10 minipreps on the overnight culture with RFP pSB4A5, according to protocol.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
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-                                    <div class="row">
-                                        <h2 class="title-style-3">6<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Stocked primers VF2 and VR: storage stock (100µM) and working stock (10µM).</p>
-                                            <p>Colony PCR of transformants from yesterday with primers VF2 and VR followed by gel electrophoresis. Gel picture yielded no bands. This might be attributed to a fault in the transformation.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
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-                                    <div class="row">
-                                        <h2 class="title-style-3">7<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Transformation of same BioBricks as on 5<sup>th</sup> July using a different transformation protocol since we believed that the negative result was due to a mistake during transformation.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">8<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Finally, the plasmids containing K1149051 and BBa_E0840 yielded a few colonies. However, the ones containing the promoters did not. Thus, we believe that the distribution kit plate containing the promoters is defective and the mKate ligation also featured some problems.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">11<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>To test whether the distribution kit might be the problem, transformation of BBa_J23113 in OneShot® TOP10 chemically competent cells (Invitrogen) to make sure the transformation process is not the cause of the negative results. Also, a plasmid containing TU Delft 2015 csgA Biobrick was used as a positive control.</p>
-                                            <p>For the mKate construct we will obtain the backbone from another construct instead of the linearized backbones from the iGem distribution kit.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">12<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Measure DNA concentration of biobricks from registry by nanodrop.</p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>Product</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>J23113</td>
-                                                        <td>89.3</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23117</td>
-                                                        <td>91.7</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23105</td>
-                                                        <td>89.3</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23108</td>
-                                                        <td>92.7</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23100</td>
-                                                        <td>93.4</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>E0840</td>
-                                                        <td>81.9</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>As can be seen from the table, there is DNA presence in the samples. Therefore, something else should be causing the transformations not to work. Since last year's distribution kit plates are still available in the lab and those plasmids were not taken we will try to get these parts from 2015 distribution kit.</p>
-                                            <p><strong>María</strong></p>
-                                            <p>Transfer colonies for K1149051 and BBa_E0840 to overnight LB+Cm culture. A colony containing CsgA was transfered as well to be able to isolate and use that plasmid as standard backbone for all synthesized parts.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">13<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Plasmid isolation of overnight cultures prepared yesterday and preparation of K1149051 and BBa_E0840 samples for sequencing.</p>
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Cryostocks of K1149051 and BBa_E0840 overnight culures. Stored at -80 degrees.</p>
-                                            <p>Digestion of backbones pSB1C3, pSB4A5 and all gBlocks with EcorI and PstI.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">14<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Gel purification of digested backbones. Dissolved in nuclease free water, stored at -20 degrees.</p>
-                                            <p>Ligation of synthesized fragments into backbones.</p>
-                                            <p><strong>María</strong></p>
-                                            <p>After all the problems transforming in the past weeks, we tested if we could use our homemade chemically competent cells for future transformations by transforming a couple of positive controls, which were taken from last year's plasmids. The transformations worked so these cells can still be used.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">15<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Transformation of Lycka's ligation products from yesterday and the BioBricks from the distribution kit were given one last chance. Transformed cells were plated after doubling their concentration.</p>
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Dissolution of promoter BioBricks from 2015 iGEM distribution kit.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">18<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Restriction of backbones pSB1C3 and pSB4A5 with EcorI and PstI. Gel electrophoresis of digested fragments. Gel picture yielded no bands.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">19<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Transformation of BioBricks from the distribution kit of 2015, since the ones from 2016 yielded no colonies. Positive control: csgA. Negative control: water. Biobricks: J23100, J23108, J23105, J23117, J23113. Overnight cultivation yielded no result, we decide to drop this and add the promoters to the parts that need it by PCR.</p>
-                                            <p><strong>María</strong></p>
-                                            <p>Restriction of pSB1C3 and pSB4A5 backbones with EcoRI and PstI</p>
-                                            <p>Ligation of synthesized parts that could not be ligated on 14<sup>th</sup> of July: INP_Sil_Sdom in pSB1C3 and BolA_ind and BolA_con in both pSB1C3 and pSB4A5.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">20<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka and Tessa</strong></p>
-                                            <p>Transformation of plasmids ligated by Maria (19<sup>th</sup> of July) together with the ones ligated on 14<sup>th</sup> of July. After overnight culture the following plates contained colonies. pSB1C3: INP_Sil_Sdom, OmpA_Sil_Taur, Sil_Sdom, SulA, BolA_ind, BolA_con, phaP. pSB4A5: BolA_con. The ones with a negative result were ligated again the next day.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">21<sup>st</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Colony PCR of yesterday's transformation results from Lycka and Tessa. However, not all transformations yielded colonies.</p>
-                                            <img src='https://static.igem.org/mediawiki/2016/1/1a/T--TU_Delft--Electrophoresis_20160721_1.png' alt='electrophoresis'>
-                                            <p>Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in selective LB overnight.</p>
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Ligation of mKate, mVenus, mCerulean, OmpA_Sil_Sdom and LacI into pSB1C3. Ligation of OmpA_Sil_Taur, OmpA_Sil_Sdom, Sil_Sdom, SulA and BolA_ind into pSB4A5. Left at room temperature overnight.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">22<sup>nd</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Continue with colony PCR of 20<sup>th</sup> of July transformation results from Lycka and Tessa.</p>
-                                            <img src='https://static.igem.org/mediawiki/2016/0/0b/T--TU_Delft--Electrophoresis_20160722.png' alt='electrophoresis'>
-                                            <p>Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in selective LB overnight.</p>
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Transformation of ligation products from yesterday. Cells containing backbones pSB1C3 or pSB4A5 were plated on plates supplemented with chloramphenicol or ampicilin, respectively. After overnight cultivation the following plates contained colonies: mKate, mVenus, mCerulean, OmpA_Sil_Sdom, Sil_Sdom, SulA, BolA_ind.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">23<sup>rd</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Continue with colony PCR of yesterday's transformation results from Lycka.</p>
-                                            <img src='https://static.igem.org/mediawiki/2016/8/8d/T--TU_Delft--Electrophoresis_20160723.png' alt='electrophoresis'>
-                                            <p>Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in selective LB overnight.</p>
-                                            <p>Pick all colonies selected by colony PCR on 21<sup>st</sup>, 22<sup>nd</sup> and 23<sup>rd</sup> of July and transfer to selective LB according to the resistance expressed by each plasmid.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">24<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Cryostock and plasmid isolation of overnight cultures prepared yesterday by Maria. Cryostocks stored at -80 degrees, isolated plasmids stored at -20 degrees.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">25<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Nanodrop and prepare for sequencing plasmids isolated yesterday.</p>
-                                            <p>Sequencing resulted in the following. Correct sequence: Sil_Sdom (pSB1C3), OmpA_Sil_Taur (pSB1C3), Sil_Sdom (pSB4A5), SulA (pSB4A5). Sequence with mutations: SulA (pSB1C3), BolA_ind (pSB1C3), J23108 (pSB1C3), BolA_con (pSB4A5), BolA_ind (pSB4A5). Different sequence: BolA_con (pSB1C3), INP_sil_Sdom (pSB1C3), OmpA_Sil_Sdom (pSB4A5). Mutated sequences can be repaired by PCR and blunt end ligation.</p>
-                                            <p><strong>María</strong></p>
-                                            <p>Prepare new digested pSB1C3 and pSB4A5 backbones by restricting with EcoRI and PstI and gel isolating the restricted product.</p>
-                                            <p>New ligation of those synthesized fragments that have not yielded any positive results (transformation or sequence confirmation) in the past week in their respective backbones.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">27<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Colony PCR of plates transformed yesterday.</p>
-                                            <img src='https://static.igem.org/mediawiki/2016/b/bf/T--TU_Delft--Electrophoresis_20160727_1.png' alt='electrophoresis'>
-                                            <img src='https://static.igem.org/mediawiki/2016/e/e2/T--TU_Delft--Electrophoresis_20160727_2.png' alt='electrophoresis'>
-                                            <p>Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated the same day in selective LB overnight.</p>
-                                            <p><strong>María and Tessa</strong></p>
-                                            <p>Make new chemically competent cells. They were tested for transformation capacity with a positive control plasmid (RFP in pSB4A5). Also, untransformed cells were plated in plates supplemented with Cm or Amp to test whether they had developed a resistance during the process.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">28<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Cryostock, plasmid isolation and preparation of samples for sequencing of the overnight cultures prepared yesterday by Lycka. Only INP_Sil_Sdom in pSB4A5 was sequenced confirmed with a mutation to be fixed by PCR. The other sequences aligned to a fragment of GFP unknown to us, we assumed it either came from a contamination of the old restriction enzymes we were using or from the pSB1C3 backbone that was taken from last year's team plasmid stock and probably was poorly labeled. Thus, we decided to use another backbone and the new enzymes sponsored to all iGEM teams by New England Biolabs.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">29<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka and Célina</strong></p>
-                                            <p>Repeat of the colony PCR of plates from the 26<sup>th</sup> of July that yielded a negative result.</p>
-                                            <img src='https://static.igem.org/mediawiki/2016/0/02/T--TU_Delft--Electrophoresis_20160729.png' alt='electrophoresis'>
-                                            <p>Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in LB and the applicable antibiotic overnight.</p>
-                                            <p>Transformation of OmpA_Sil_Sdom in both different backbones.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">30<sup>th</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Colony PCR of OmpA_Sil_Sdom in both backbones from the 29<sup>th</sup> of July. The PCR machine was filled with colonies from older plates which had not yielded any good colonies yet: mKate (26<sup>th</sup> of July), mKate (mKate 30<sup>th</sup> of June), INP_Sil_Sdom (20<sup>th</sup> of July).</p>
-                                            <img src='https://static.igem.org/mediawiki/2016/2/2f/T--TU_Delft--Electrophoresis_20160730.png' alt='electrophoresis'>
-                                            <p>Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in LB and the applicable antibiotic overnight.</p>
-                                            <p>Cryostocking and plasmid isolation of colonies picked by on 29<sup>th</sup> of July. Cryostocks stored at -80 degrees, isolated plasmid stored at -20 degrees.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">31<sup>st</sup> July 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Cryostock and plasmid isolation of the overnight cultures prepared yesterday by Lycka and preparation of samples for sequencing of plasmids isolated yesterday and today. Sequence of mCerulean was confirmed (although it had a silent mutation). OmpA_Sil_Sdom in pSB4A5 and INP_Sil_Sdom in pSB1C3 had a deletion to be fixed by PCR. The rest either of sequences contained the random GFP sequenced mentioned before (since they are old ligation products) or multiple mutations that could not be fixed by PCR.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <a href="#notes" class="btn btn-info" role="button" onclick="$('html,body').animate({scrollTop: $('#notes').offset().top}, 'slow');" style="text-decoration:none; color:#f3f4f4; float:right;">Back to Top</a>
-                                </div>
-                            </div>
-                        </div>
-                        <div class="panel panel-default">
-                            <a class="collapsed" data-toggle="collapse" data-parent="#accordion" href="#collapseThree" aria-expanded="false" aria-controls="collapseThree">
-                                <div class="panel-heading" role="tab" id="headingThree">
-                                    <h4 class="panel-title">
-                                        August
-                                    </h4>
-                                </div>
-                            </a>
-                            <div id="collapseThree" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingThree">
-                                <div class="panel-body">
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-                                    <div class="row">
-                                        <h2 class="title-style-3">2<sup>nd</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Amplified E0840 (GFP) out of a pSB1C3-GFP plasmid using Phusion PCR. Four reactions of 50µl.</p>
-                                            <p>Nanodropped PCR products.</p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>Product</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>E0840</td>
-                                                        <td>339.6</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>E0840</td>
-                                                        <td>554.1</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>E0840</td>
-                                                        <td>596.9</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>E0840</td>
-                                                        <td>567.6</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>Ran a 1% agarose gel of the PCR product to verify product size.</p>
-                                            <img src="https://static.igem.org/mediawiki/2016/9/98/T--TU_Delft--Electrophoresis_20160802.png" alt="electrophoresis">
-                                            <p>Stored product 2 and 4 in the fridge for later use.</p>
-                                            <p><strong>Lara</strong></p>
-                                            <p>Digested the synthesized parts LacI, phaP, mKate, the backbones pSB1C3 and pSB4A5 and the Biobrick K1149051 with EcoRI and PstI. The restriction products were run over a 1% agarosegel and subsequently gel-purified. </p>
-                                            <p>Concentrations of purified DNA products.</p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>DNA construct</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>LacI</td>
-                                                        <td>15.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>phaP</td>
-                                                        <td>8.2</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mKate</td>
-                                                        <td>8.7</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>K1149051</td>
-                                                        <td>13.8</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>pSB1C3</td>
-                                                        <td>25.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>pSB1C3</td>
-                                                        <td>13.1</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>pSB1C3</td>
-                                                        <td>9.3</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>pSB4A5</td>
-                                                        <td>10.4</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>pSB4A5</td>
-                                                        <td>7.3</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>pSB4A5</td>
-                                                        <td>6.4</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                        </div>
-                                    </div>
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">3<sup>rd</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lara</strong></p>
-                                            <p>Ligated the synthesized parts that were restricted and gel-purified yesterday and the backbones. The following ligation products were formed: LacI-pSB1C3, phaP-pSB1C3, mKate-pSB1C3, LacI-pSB4A5 and K1149051-pSB4A5. The ligation protocol was used and the below stated lengths for the vector and the inserts were used. </p>
-                                            <h4>Lengths of inserts and backbones.</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>DNA construct</th>
-                                                <th>DNA length (bp)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>LacI</td>
-                                                        <td>1335</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>phaP</td>
-                                                        <td>648</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mKate</td>
-                                                        <td>790</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>K1149051</td>
-                                                        <td>4271</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>pSB1C3</td>
-                                                        <td>2070</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>pSB4A5</td>
-                                                        <td>3395</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>Transformations were performed with the just obtained ligation products and additionally the previously obtained mVenus-pSB1C3 was also transformed into TOP10 cells. A DNA amount of 2 µL was used; as positive control for the ampicilline plates the plasmid RFP-pSB4A5 was used during transformation and for the chloramphenicol plates the plasmid RFP-pSB1C3 was used. Transformed cells were plated on selective LB-agar plates and incubated overnight at 37 degrees Celsius. </p>
-                                        </div>
-                                    </div>
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">4<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lara</strong></p>
-                                            <p>Performed a DreamTaq colony PCR for yesterday's transformations; eight colonies per plate were picked and analyzed. </p>
-                                            <p>Colonies were transferred into selective liquid LB-medium.</p>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>DNA construct transformation</th>
-                                                <th>Selective medium</th>
-                                                <th>Colonies picked</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>K1149051-pSB4A5</td>
-                                                        <td>LB-Amp</td>
-                                                        <td>2</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>phaP-pSB1C3</td>
-                                                        <td>LB-Cm</td>
-                                                        <td>19, 20 and 21</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mKate-pSB1C3</td>
-                                                        <td>LB-Cm</td>
-                                                        <td>31</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mVenus-pSB1C3</td>
-                                                        <td>LB-Cm</td>
-                                                        <td>36, 40 and 41</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>LacI-pSB1C3</td>
-                                                        <td>LB-Cm</td>
-                                                        <td>46, 48 and 49</td>
-                                                </tbody>
-                                            </table>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">5<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lara</strong></p>
-                                            <p>Some of the overnight cultures were opened inside the shaker, so these were not sterile anymore. Colonies 21 (phaP-pSB1C3) and 47 (LacI-pSB1C3) had to be picked and transferred into liquid LB-medium again. </p>
-                                            <p>From the other overnight cultures, plasmids were isolated using Miniprep. </p>
-                                            <h4>Concentrations of plasmid isolations. </h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>DNA construct</th>
-                                                <th>Colony number </th>
-                                                <th>Concentration (ng/L)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>K1149051-pSB4A5</td>
-                                                        <td>2</td>
-                                                        <td>271.7</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>phaP-pSB1C3</td>
-                                                        <td>19</td>
-                                                        <td>398.0</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>phaP-pSB1C3</td>
-                                                        <td>20</td>
-                                                        <td>207.1</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mKate-pSB1C3</td>
-                                                        <td>31</td>
-                                                        <td>83.3</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mVenus-pSB1C3</td>
-                                                        <td>36</td>
-                                                        <td>142.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mVenus-pSB1C3</td>
-                                                        <td>40</td>
-                                                        <td>306.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mVenus-pSB1C3</td>
-                                                        <td>41</td>
-                                                        <td>65.3</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>These DNA samples were sent to sequencing.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">9<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Charlotte</strong></p>
-                                            <p>Started correcting point mutations using PCR in BioBricks that had a non-silent mutation in a coding area. The plasmids being corrected are SulA (pSB1C3), BolA_ind (pSB1C3), BolA_con (pSB4A5) and INP_Sil_Sdom (pSB4A5). Primers were ordered by María. Primers were first diluted for storage (100 µM) and working stock (10 µM). The concentrations of the plasmids with point mutations were determined using a Nanodrop.  </p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>BioBrick</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>SulA (pSB1C3)</td>
-                                                        <td>167.7</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>BolA_ind (pSB1C3)</td>
-                                                        <td>109.1</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>BolA_con (pSB4A5)</td>
-                                                        <td>130.4</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>INP_Sil_Sdom (pSB4A5)</td>
-                                                        <td>166.7</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>The PCR to correct the mutations was done according to the Phusion HF protocol. The elongation time was set at 75 seconds, and the annealing time was set at 57ºC. The PCR product was loaded on a 1% TAE agarose gel for electrophoresis, in order to determine of the PCR worked.  </p>
-                                            <img src='https://static.igem.org/mediawiki/2016/5/50/T--TU_Delft--File_20160809Gel_mut1.jpg' alt='electrophoresis'>
-                                            <p>Left is a simulated image by SnapGene, right the actual gel picture. The lane marked with L is the Promega 1kb ladder, lane 1 contains SulA (pSB1C3), lane 2 contains BolA_ind (pSB1C3), lane 3 contains BolA_con (pSB4A5) and lane 4 contains INP_Sil_Sdom (pSB4A5) </p>
-                                            <p>The bands for BolA_ind, BolA_con and INP_Sil_Sdom are correct (lanes 2-4). The band for SulA (lane 1) is not as expected. This sample is checked on gel again. <p>
-                                                <img src='https://static.igem.org/mediawiki/2016/7/7a/T--TU_Delft--PCRmut2.jpg' alt='electrophoresis'>
-                                            <p> Left is a simulated image by SnapGene, right the actual gel picture. The lane marked with L is the Promega 1kb ladder, lane 1 contains SulA (pSB1C3. The other bands contain result from another experiment, irrelevant for this experiment.</p>
-                                            <p>The result from the second electrophoresis of SulA is still not as expected, we will start over with the correction of this BioBrick later. For now, we continue with BolA_ind (pSB1C3), BolA_con (pSB4A5) and INP_Sil_Sdom (pSB4A5).</p>
-                                            <p> BolA_ind (pSB1C3), BolA_con (pSB4A5) and INP_Sil_Sdom (pSB4A5) wetre purified according to protocol. The obtained concentrations were measured using Nanodrop.
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>BioBrick</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>BolA_ind (pSB1C3)</td>
-                                                        <td>99.9</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>BolA_con (pSB4A5)</td>
-                                                        <td>98.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>INP_Sil_Sdom (pSB4A5)</td>
-                                                        <td>61.5</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p> The purified PCR product was restricted with DpnI, to restrict all methylated DNA containing the point mutation, leaving us with the corrected DNA. Restriction was done according to protocol. After 1 hour of incubation at 37ºC the restriction products were purified according to protocol. The prified restriction products were stored at -20ºC.</p>
-                                        </div>
-                                    </div>
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">10<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Restricted PCR product of 3rd August, J23100 E0840, J23113 E0840 and J23117 E0840, with EcoRI-HF and PstI. </p>
-                                            <p>Purified restriction product.</p>
-                                            <p>Nanodropped purified product.</p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>Product</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>J23100 E0840</td>
-                                                        <td>14.7</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23113 E0840</td>
-                                                        <td>21.3</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23117 E0840</td>
-                                                        <td>23.3</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>Ligated purified product into pSB1C3.</p>
-                                            <p>Transformed ligation product into TOP10 strain. Using RFP as positive control and sterile MilliQ as negative control. Plated on plates with LB agar and CM.</p>
-                                            <p><strong>Lara</strong></p>
-                                            <p>Of the previously sent DNA samples to sequencing, mVenus-pSB1C3, mKate-pSB1C3 and phaP-pSB1C3 were sequence confirmed. LacI-pSB1C3 and K1149051 were analysed again. Since the previous selected colonies for LacI-pSB1C3 turned out to contain the construct of the positive control RFP, a new colony-PCR was performed. Besides that, colony 47 of August 4<sup>th</sup> was also transferred into liquid medium. </p>
-                                            <p>Via Phusion-PCR, the promoters J23100, J23105, J23108, J23113 and J23117 were added to the yesterday sequence confirmed phaP-pSB1C3. The melting temperature of the designed primers were for all 58 degrees Celsius; the template concentration was 102.0 ng/µl and the final construct should obtain 835 bp. The Phusion-PCR ran overnight.</p>
-                                            <p><strong>Charlotte</strong></p>
-                                            The concentrations of the previously purified restriction products were measured using a Nanodrop:</p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>Product</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>BolA_ind (pSB1C3)</td>
-                                                        <td>16.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>BolA_con (pSB4A5)</td>
-                                                        <td>14.4</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>INP_Sil_Sdom (pSB4A5)</td>
-                                                        <td>9.5</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p> The restriction products were blunt-end ligated in order to circularize the plasmids. Since our ligation tool does not calculate the concentrations needed for blunt-end ligation, the following protocol was used:</p>
-                                            <h4>Blunt-end ligation protocol</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>Component</th>
-                                                <th>Amount</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>T4 ligation buffer (10X)</td>
-                                                        <td>2 µL</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>DNA</td>
-                                                        <td>~80 ng</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>T4 Ligase</td>
-                                                        <td>1 µL</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Nuclease-free water</td>
-                                                        <td>up to 20 µL</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p> The T4 ligase was added last, the ligation mixture was left to incubate for 4 hours. After incubation, the ligation product was transformed into homemade chemically competent TOP10 cells, according to protocol. The tranformations were set to incubate overnight at 37ºC</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">11<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Colony PCR'd colonies from yesterdays transformation using Q5<sup>&reg</sup> mastermix.</p>
-                                            <p>Ran a 1% agarose gel of the PCR product.</p>
-                                            <img src='https://static.igem.org/mediawiki/2016/d/d5/T--TU_Delft--Electrophoresis_20160811.png' alt='electrophoresis'>
-                                            <p> L is the Promega 1kb ladder, 1-6 is J23100 E0840, 7-14 is J23113 E0840 and 15-22 is J23117 E0840.
-                                            <p>Transferred colony 14 (J23113 E0840 pSB1C3) and 21 (J23117 E0840 pSB1C3) into liquid LB.<p>
-                                            <p><strong>Lara</strong></p>
-                                            <p>Plasmids were isolated from the colonies that were grown overnight.</p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>DNA construct</th>
-                                                <th>Colony number</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>LacI-pSB1C3</td>
-                                                        <td>47</td>
-                                                        <td>56.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>LacI-pSB1C3</td>
-                                                        <td>51</td>
-                                                        <td>86.4</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23117 E0840</td>
-                                                        <td>54</td>
-                                                        <td>141.4</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>LacI-pSB1C3 was sent to sequencing.</p>
-                                            <p><strong>Charlotte</strong></p>
-                                            <p>Picked 8 colonies for the plates containing cells tranformed with BolA_ind (psB1C3) and 8 for BolA_con (pSB4A5). The colonies on the plate containing cells transformed with INP_Sil_Sdom were still too small to pick so they were left to incubate at 37ºC a little longer before picking 8 colonies for colony PCR. </p>
-                                            <p> The colony PCR was done according to protocol, using DreamTaq polymerase. The PCR products were loaded on a 1% agarose gel for electrophoresis. Colony 16 didn't fit in the PCR machine anymore, so this colony is also not loaded on the gel.</p>
-                                            <img src='https://static.igem.org/mediawiki/2016/0/09/T--TU_Delft--Charlotte_ColPCR1.jpg' alt='electrophoresis'>
-                                            <p>Left is a simulated image by SnapGene, right the actual gel picture. For BolA_ind, colonies 2,3 and 4 had a band at the correct height. For BolA_con, colonies 11, 12 and 13 had a band at the correct height. For INP_Sil_Sdom, colonies 17, 19 and 20 were correct. These colonies were transferred to selective liquid LB and grown overnight in a shaker at 37ºC and 250 rpm. </p>
-                                            <p><strong>Charlotte & Liza</strong></p>
-                                            <p> Tested 15 µm fluorescent polystyrene beads in the MicroTime 200 confocal microscope to see if the microscope is able to establish lasing in a bead. We used a 1 mW laser with an excitation wavelength of 532 nm. The bead was imaged at different intensities to see if we were able to see a shift to lasing at higher excitation energies.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">12<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Miniprepped colony 14 and 21 of yesterdays overnight culture.</p>
-                                            <p>Nanodropped miniprep product.</p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>Product</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>J23113 E0840 in pSB1C3</td>
-                                                        <td>140.6</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23117 E0840 in pSB1C3</td>
-                                                        <td>325.6</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>Cryostocked colony 14 and 21.</p>
-                                            <p>Send miniprep product for sequencing. [Sequence Confirmed]</p>
-                                            <p><strong>Lara</strong></p>
-                                            <p>The results for the sequencing of LacI-pSB1C3 were checked: For both colonies 47 and 51 there was a 124 bp long exact same insert in the middle of the LacI-gene. In colony 54, there were two point mutations. The first one at location 2215, changing an Arg into Leu; the second mutation was on location 2625 changing a Glu into a stopcodon. </p>
-                                            <p>These two point mutations need to be fixed. Since normal mutation fixing by PCR only handles one mutation at a time, a new strategy was thought of: Two different linear fragments should be made, both fixing one mutation and able to join together with the other part via Gibson Assembly. The primers were designed and ordered.</p>
-                                            <p>The Phusion-PCR products of the different promoters that were obtained were transformed into TOP10 cells. For this, 2µl DNA was used per transformation, RFP-pSB1C3 as a positive control and MilliQ as a negative control. All transformations were plated onto LB-Cm plates and stored at 37 degrees Celsius. </p>
-                                            <p><strong>Charlotte</strong></p>
-                                            <p> The DNA of the colonies that were grown overnight was isolated using miniprep according to protocol. The isolated DNA was stored at -20ºC. The remaining culture was cryostocked according to protocol and stored at -80ºC.</p>
-                                            <p><strong>Charlotte & Liza</strong></p>
-                                            <p> Did a second attempt to get lasing in a bead with a confocal microscope.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!--put an activtiy here -->
-                                    <div class="row">
-                                        <h2 class="title-style3">13<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lara</strong></p>
-                                            <p>A colony PCR was performed for the transformations of the different promoters attached to phaP-pSB1C3. The expected amplification length is approximately 1kb. Four colonies per plate were analyzed in the colony PCR. </p>
-                                            <p>This gel showed that only colony 35 (lane 15) did not contain the right PCR-product. The other colonies were transferred to liquid LB-Cm and cultivated overnight at 37 degrees Celsius.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity -->
-                                    <!--put an activtiy here -->
-                                    <div class="row">
-                                        <h2 class="title-style3">14<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lara</strong></p>
-                                            <p>The overnight grown cultures of the different promoters attached to phaP-pSB1C3 were plasmid isolated. </p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>DNA construct</th>
-                                                <th>Colony number</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>J23100 phaP pSB1C3</td>
-                                                        <td>33</td>
-                                                        <td>83.1</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23100 phaP pSB1C3</td>
-                                                        <td>34</td>
-                                                        <td>80.4</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23105 phaP pSB1C3</td>
-                                                        <td>25</td>
-                                                        <td>101.6</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23105 phaP pSB1C3</td>
-                                                        <td>26</td>
-                                                        <td>113.0</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23105 phaP pSB1C3</td>
-                                                        <td>27</td>
-                                                        <td>97.4</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23108 phaP pSB1C3</td>
-                                                        <td>18</td>
-                                                        <td>93.4</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23108 phaP pSB1C3</td>
-                                                        <td>19</td>
-                                                        <td>120.0</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23108 phaP pSB1C3</td>
-                                                        <td>20</td>
-                                                        <td>100.0</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23113 phaP pSB1C3</td>
-                                                        <td>9</td>
-                                                        <td>94.3</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23113 phaP pSB1C3</td>
-                                                        <td>10</td>
-                                                        <td>85.0</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23113 phaP pSB1C3</td>
-                                                        <td>11</td>
-                                                        <td>110.0</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23117 phaP pSB1C3</td>
-                                                        <td>1</td>
-                                                        <td>101.2</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23117 phaP pSB1C3</td>
-                                                        <td>2</td>
-                                                        <td>100.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>J23117 phaP pSB1C3</td>
-                                                        <td>3</td>
-                                                        <td>110.0</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>These plasmids were sent to sequencing.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity -->
-                                    <!--put an activtiy here -->
-                                    <div class="row">
-                                        <h2 class="title-style3">13<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lara</strong></p>
-                                            <p>A colony PCR was performed for the transformations of the different promoters attached to phaP-pSB1C3. The expected amplification length is approximately 1kb. Four colonies per plate were analyzed in the colony PCR. </p>
-                                            <p>This gel showed that only colony 35 (lane 15) did not contain the right PCR-product. The other colonies were transferred to liquid LB-Cm and cultivated overnight at 37 degrees Celsius.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity -->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">15<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Charlotte</strong></p>
-                                            The concentrations of the previously isolated DNA was checked on Nanodrop. The following concentrations were measured:
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>BioBrick</th>
-                                                <th>Colony number</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td scope="row" rowspan="3">BolA_ind (pSB1C3)</td>
-                                                        <td>2</td>
-                                                        <td>63.0</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>3</td>
-                                                        <td>61.8</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>4</td>
-                                                        <td>68.3</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td scope="row" rowspan="3">BolA_con (pSB4A5)</td>
-                                                        <td>11</td>
-                                                        <td>101.9</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>12</td>
-                                                        <td>71.7</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>14</td>
-                                                        <td>90.9</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td scope="row" rowspan="3">INP_Sil_Sdom (pSB4A5)</td>
-                                                        <td>17</td>
-                                                        <td>88.0</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>19</td>
-                                                        <td>74.4</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>20</td>
-                                                        <td>43.2</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p> The isolated DNA was prepared and sent for sequencing, according to protocol. </p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!--put an activtiy here -->
-                                    <div class="row">
-                                        <h2 class="title-style3">16<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Charlotte</strong></p>
-                                            <p>Sequencing had the following results:</p>
-                                            <li><b>BolA_ind (pSB1C3):</b> All colonies had large inserts or deletions, so they couldn't be used for further experiments. We will try to repair the mutations again later. </li>
-                                            <li><b>BolA_con (pSB4A5):</b> Colony 14 has an insert and colony 11 and 12 have a small gap. For colony 11 this is probably a readout error, when you look closer to the chromatogram. We can use colony 11 for further experiments.</li>
-                                            <li><b>INP_Sil_Sdom (pSB4A5):</b> Colony 17 has a nonsilent mutation in the promoter and colony 20 has a gap in the promoter. Colony 19 has one silent mutation and one nonsilent mutation, but in the linker region. We can use colony 19 for further experiments.</li>
-                                        </div>
-                                    </div>
-                                    <!-- end activity -->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">17<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Charlotte & Liza</strong></p>
-                                            <p> Did a third attempt to get lasing in a bead with a confocal microscope.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">18<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Transformation of the InterLab Study plasmids into BL21.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">19<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Only Test Device 2 and 3 and positive control have yielded (very little) colonies. Decided to try and tranform them into Top10.</p>
-                                            <p><strong>Lara</strong></p>
-                                            <p>A colony PCR was performed for the transformation of LacI-pSB1C3.</p>
-                                            <p>Since none of the colonies yielded a positive result in the colony-PCR, the colony-PCR was performed again. This time with a new protocol, first boiling the colonies and using GoTaq buffer. </p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">22<sup>nd</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Transformation of the InterLab Study plasmids into Top10.</p>
-                                            <p>Colony PCR of J23100 E0840 pSB1C3 using GoTaq<sup>&reg</sup>. Plate from 11th August.</p>
-                                            <p>Ran a 1% agarose gel to verify part size</p>
-                                            <img src='https://static.igem.org/mediawiki/2016/a/a1/T--TU_Delft--Electrophoresis_20160822.png' alt='electrophoresis'>
-                                            <p>L is the Promega 1kb ladder, 1-9 are J23100 E0840.</p>
-                                            <p>Colony 1,2 and 3 were put in liquid LB+CM and incubated overnight.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">23<sup>rd</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Colony PCR of the InterLab Study plates using DreamTaq<sup>&reg</sup>. Picked eight colonies of each plate.</p>
-                                            <p>Cryostocked J23100 E0840 pSB1C3 in Top10.</p>
-                                            <p>Ran a 1% agarose gel of the InterLab Study colony PCR products.</p>
-                                            <img src='https://static.igem.org/mediawiki/2016/4/46/T--TU_Delft--Electrophoresis_20160823.png' alt='electrophoresis'>
-                                            <p>L is the Promega 1kb ladder, 1-8 is Test Device 1, 9-16 is Test Device 2, 17-24 is Test Device 3, 25-32 is Negative Control and 33-40 is Positive control.</p>
-                                            <p>Colony 1, 2, 4, 9-11, 17-19, 26, 27, 29 and 33-36 were picked for overnight culture in liquid LB+CM.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">24<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Put InterLab Study cultures in fridge for later use.</p>
-                                            <p><strong>Charlotte</strong></p>
-                                            <p>Started a second round of correcting mutations in our designed BioBricks using PCR. The BioBricks that will be corrected are SulA (pSB1C3) BolA_ind (pSB1C3), INP_Sil_Sdom (pSB1C3) and OmpA_Sil_Sdom (pSB4A5). Prior to starting the correction of the mutations using PCR, the concentrations of these BIoBricks were measured on the Nanodrop:</p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>BioBrick</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>SulA (pSB1C3)</td>
-                                                        <td>167.6</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>BolA_ind (pSB1C3)</td>
-                                                        <td>109.1</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>INP_Sil_Sdom (pSB1C3)</td>
-                                                        <td>139.2</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>OmpA_Sil_Sdom</td>
-                                                        <td>170.8</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p> The primers were diluted according to the manufacturers' instructions to a stock of 100mM. These stocks were consequently 10 times diluted to a working stock of 10mM. </p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">25<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Resuspended FITC according to InterLab protocol.</p>
-                                            <p><strong>Charlotte</strong></p>
-                                            <p>Ran the PCR to fix the mutations in the BioBricks according to the Phusion HF PCR protocol, with an annealing time of 57ºC and and elongation time of 1.5 minutes. The results of the PCR were confirmed on gel, according to the electrophoresis protocol. </p>
-                                            <img src='https://static.igem.org/mediawiki/2016/8/86/T--TU_Delft--Charlotte_gel3.png' alt='electrophoresis'>
-                                            <p>Left is a simulated image by SnapGene, right the actual gel picture. The lane marked with L is the Promega 1kb ladder, lane 1 contains SulA (pSB1C3), lane 2 contains BolA_ind (pSB1C3), lane 3 contains INP_Sil_Sdom (pSB1C3) and lane 4 OmpA_Sil_Sdom (pSB4A5) </p>
-                                            <p> The bands from BolA_ind (pSB1C3), INP_Sil_Sdom (pSB1C3) and OmpA_Sil_Sdom (pSB4A5) are at the correct height compared to the simulation. The lane containing SulA shows the same result as the previous attempt to correct this BoBrick. PRobably something goed wrong in the PCR, it's likely that the primers anneal at the worng position. We will not continue this BioBrick and use SulAin another backbone. The other PCR products were purified according to protocol and the concentrations were measured using Nanodrop. </p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>BioBrick</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>BolA_ind (pSB1C3)</td>
-                                                        <td>107.7</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>INP_Sil_Sdom (pSB1C3)</td>
-                                                        <td>88.7</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>OmpA_Sil_Sdom</td>
-                                                        <td>100.3</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p> The purified PCR products wererestricted with DpnI to remove any methylated and thus mutated DNA, according to the restriction protocol. Since our heat block was broken the samples were incubated in the stove at 37ºC. The restriction productswere purified according to protocol and stored at -20ºC.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">29<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Transformed J23100-, J23105-, J23108-, J23113- and J23117 E0840 pSB1C3, and BolA_con pSB4A5 into BL21.</p>
-                                            <p><strong>Charlotte</strong></p>
-                                            <p>Checked the concentrations of the purified restriction products on Nanodrop:</p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>BioBrick</th>
-                                                <th>Concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>BolA_ind (pSB1C3)</td>
-                                                        <td>14.7</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>INP_Sil_Sdom (pSB1C3)</td>
-                                                        <td>11.9</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>OmpA_Sil_Sdom</td>
-                                                        <td>12.6</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p> The restriction products were blunt-end ligated according to the ligation protocol. The ligated BioBricks were transformed into homemade TOP10 cells, according to protocol. For the controls only 1/3 of the colume was used due to high transformation efficiency observed before. The transformants were plated on selective plated and left to incubate overnight at 37ºC.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">30<sup>th</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>PCR lineralization of vectors and inserts for gibson assembly pha operon (BBa_K1149051) to fluorophores (GFP, mVenus, mKate, mCerulean). Phusion polymerase. Annealing temperature 60 degrees, elongation time 2 minutes.</p>
-                                        </div>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Did the InterLab Study plate reader measurements according to the InterLab plate reader protocol.</p>
-                                            <p>Streaked J23105- and J23117 E0840 pSB1C3 out on new plates, because they had too many colonies.</p>
-                                            <p>Colony PCR'd six colonies of BolA_con, J23100 E0840, J323108 E0840 and J23113 E0840 using GoTaq<sup>&reg</sup>.</p>
-                                            <p>Ran a 1% agarose gel of PCR products to verify part size.</p>
-                                            <img src='https://static.igem.org/mediawiki/2016/3/39/T--TU_Delft--Electrophoresis_20160830.png' alt='electrophoresis'>
-                                            <p>L is the Promega 1kb ladder, 1-6 is BolA_con, 7-12 is J23100 E0840, 12-18 is J23108 E0840 and 19-24 is J23113 E0840.</p>
-                                            <p>Picked colony 1, 6, 11, 13, 18, 20 and 21 for overnight culture.</p>
-                                            <p><strong>Charlotte</strong></p>
-                                            <p> Of each transformation plate, 8 colonies were picked: Colonies 1-8 were of BolA_ind, colonies 9-16 of OmpA_Sil_Sdom and colonies 17-24 of INP_Sil_Sdom. A colony PCR was performed with the picked colonies, according to the GoTaq Colony PCR protocol. The annealing time was 1.5 minutes.</p>
-                                            <p> The PCR products were checked on gel according to the Electrophoresis protocol, which yielded the following gel: </p>
-                                            <img src='https://static.igem.org/mediawiki/2016/b/be/T--TU_Delft--ColonyPCR_CCK.png' alt='electrophoresis'>
-                                            <p>L is the Promega 1kb ladder, 1-8 is BolA_ind, 9-16 is OmpA_Sil_Sdom, 17-24 is INP_Sil_Sdom </p>
-                                            <p> Colonies 1, 3, 4, 14, 15, 16, 20, 21 and 22 were picked and transferred to selective medium for an overnight culture, according to protocol. </p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">31<sup>st</sup> August 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Electrophoresis of PCR fragments yesterday.</p>
-                                            <img src="https://static.igem.org/mediawiki/2016/7/79/T--TU_Delft--Electrophoresis_20160831_1.png" alt="electrophoresis">
-                                            <p>Picture on the left shows simulated agarose gel. Picture on the right shows the actual agarose gel. 1 - 4: vectors for mCerulean, mKate, mVenus and GFP, respectively. 5 - 6 inserts for mVenus and GFP. Gel did not yield bands at the right height. PCR is repeated at an annealing temperature of 57 degrees.</p>
-                                            <img src="https://static.igem.org/mediawiki/2016/9/9a/T--TU_Delft--Electrophoresis_20160831.png" alt="electrophoresis">
-                                            <p>Second gel also did not yield the right bands. PCR repeated overnights by María at Tm 64 degrees and elongation time 3.5 minutes in the presence of DMSO.</p>
-                                            <p>Transfer from cryostock to plate BL21 strains with fluorophores expressed. On LB agar supplemented with chloramphenicol.</p>
-                                        </div>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Cryostocked J23100-, J23108- and J23117 E0840 pSB1C3 in BL21. BolA_con didn't grow.</p>
-                                            <p>Cryostocked K1149051 pSB4A5 in BL21.</p>
-                                        </div>
-                                        <div class="col-md-12">
-                                            <p><strong>Charlotte</strong></p>
-                                            <p>The overnight cultures of BolA_ind (pSB1C3), OmpA_Sil_Sdom (pSB4A5) and INP_Sil_Sdom (pSB1C3) were cryostocked according to protocol, 3 tubes per culture. The remaining culture was used for DNA isolation by Miniprep, according to protocol. Two tubes per colony were prepared. The concentration of the isolated DNA was measured using Nanodrop, the following concentrations were measured:</p>
-                                            <h4>Nanodrop</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>BioBrick</th>
-                                                <th>Colony number</th>
-                                                <th>Tube 1 concentration (ng/µl)</th>
-                                                <th>Tube 2 concentration (ng/µl)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td scope="row" rowspan="3">BolA_ind (pSB1C3)</td>
-                                                        <td>1</td>
-                                                        <td>91.2</td>
-                                                        <td>113.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>3</td>
-                                                        <td>87.9</td>
-                                                        <td>79.6</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>4</td>
-                                                        <td>104.5</td>
-                                                        <td>87.7</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td scope="row" rowspan="3">OmpA_Sil_Sdom (pSB4A5)</td>
-                                                        <td>14</td>
-                                                        <td>133.5</td>
-                                                        <td>143.7</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>15</td>
-                                                        <td>152.5</td>
-                                                        <td>141.4</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>16</td>
-                                                        <td>171.1</td>
-                                                        <td>159.4</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td scope="row" rowspan="3">INP_Sil_Sdom (pSB4A5)</td>
-                                                        <td>20</td>
-                                                        <td>109.6</td>
-                                                        <td>90.6</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>21</td>
-                                                        <td>112.2</td>
-                                                        <td>101.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>22</td>
-                                                        <td>112.0</td>
-                                                        <td>111.9</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p> The isolated DNA was prepared and sent for sequencing, according to protocol. </p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <a href="#notes" class="btn btn-info" role="button" onclick="$('html,body').animate({scrollTop: $('#notes').offset().top}, 'slow');" style="text-decoration:none; color:#f3f4f4; float:right;">Back to Top</a>
-                                </div>
-                            </div>
-                        </div>
-                        <div class="panel panel-default">
-                            <a class="collapsed" data-toggle="collapse" data-parent="#accordion" href="#collapseFour" aria-expanded="false" aria-controls="collapseFour">
-                                <div class="panel-heading" role="tab" id="headingFour">
-                                    <h4 class="panel-title">
-                                        September
-                                    </h4>
-                                </div>
-                            </a>
-                            <div id="collapseFour" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingFour">
-                                <div class="panel-body">
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">1<sup>st</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>PCR with new primers of those parts that need to be expressed under the lac promoter. These primers will allow cloning on Addgene plasmids from the pBb series. The plasmids chosen (pBbS5a and pBbA5c) express lacI and the cloning site is placed downstream of a lac promoter. The parts to be amplified are:<br>1. BolA<br>2. INP_Sil_Sdom<br>3. OmpA_Sil_Sdom<br>4. OmpA_Sill_Taur<br>5. Sil_Sdom<br>6. SulA</p>
-                                            <img src="https://static.igem.org/mediawiki/2016/9/91/T--TU_Delft--Electrophoresis_20160901_2.png" alt="electrophoresis">
-<p> </p>
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Electrophoresis of overnight PCR for lineralization.</p>
-                                            <img src="https://static.igem.org/mediawiki/2016/2/2a/T--TU_Delft--Electrophoresis_20160901.png" alt="electrophoresis">
-                                            <p>Picture on the left shows simulated agarose gel. Picture on the right shows the actual agarose gel. 1 - 4: vectors for mCerulean, mKate, mVenus and GFP, respectively. 5 - 6 inserts for mVenus and GFP. For all lanes except lane 5, the right bands are present. Also many other bands were present so the product was gel purified.</p>
-                                            <p>Transfered one colony of BL21 on each plate from yesterday in a 50mL falcon tube with 10mL filter sterilized M9 medium with glucose supplemented with chloramphenicol.</p>
-                                        </div>
-                                        <div class='col-md-12'>
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Put three colonies of J23100-, J23105- and J23117 E0840 pSB1C3 in BL21 into liquid LB+CM.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">2<sup>nd</sup> September 2016</h2>
-                                        <div class='col-md-12'>
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Cryostocked J23100-, J23105- and J23117 E0840 pSB1C3 in BL21.</p>
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Nanodrop gel purified PCR fragments from yesterday. Concentrations are too low to work with. Repeat PCR under the same conditions, but with double volume (100µL). Done by María.</p>
-                                            <p>Measure OD600 of cultivated M9 tubes. </p>
-                                            <h4>OD600 measurements</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>Genotype</th>
-                                                <th>OD600</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>GFP J23108</td>
-                                                        <td>1.24</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mVenus</td>
-                                                        <td>0.04</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mKate</td>
-                                                        <td>0.20</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mCerulean</td>
-                                                        <td>0.03</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>The strains containing GFP and mKate grew really well, the ones containing mVenus and mCerulean did not. All of the tubes were put back in the incubator. The one with GFP was diluted 10 times in M9 medium, the one with mKate was diluted 2 times. Aliquots of 150µL were loeaded in an 96 well plate and measured in a plate reader at 37 degrees for 7 hours.</p>
-                                            <p><strong>María</strong></p>
-                                            <p>PCR of same samples as Lycka in a reaction volume of 100µL. After gel isolation and purification only for mCerulean and mKate good concentrations were reached (160 and 260 ng/µL, respectively), for the rest of samples either no bands were seen in the agarose gel or the concentration was too low again.</p>
-                                            <p><strong>Charlotte</strong></p>
-                                            <p> Analyzed sequencing results. Both the colonies of BolA_ind and OmpA_Sil_Sdom had an insert around the mutation spot, so sequences were not correct. Also all colinies of INP_Sil_Sdom had non-silent mutations. </p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
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-                                    <div class="row">
-                                        <h2 class="title-style-3">3<sup>rd</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>The cultures in M9 grew well in the incubator until an OD of approximately 1. Samples were diluted 10 times in M9 supplemented with chloramphenicol and measured in the plate reader.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">6<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka & Lara</strong></p>
-                                            <p>Colony PCR for the transformants of the Gibson Assembly. </p>
-                                            <img src="https://static.igem.org/mediawiki/2016/b/b0/T--TU_Delft--Electrophoresis_20160906.png" alt="electrophoresis">
-                                            <p>Most colonies appear to be false positives, since the bands correspond to the length of the vector only. Colonies 43 and 44 might contain the desired plasmid (with the PHB operon fused to mKate), so these are transfered to LB medium.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">8<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Miniprep, cryostock and prepare for sequencing colonies 43 and 44. The concentrations were 252.6 and 242.6 ng/µl, respectively.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
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-                                    <div class="row">
-                                        <h2 class="title-style-3">9<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>In order for the cells to grow better, we prepared enriched M9 medium (eM9), instead of regular M9.
-                                                The medium was supplemented with chloramphenicol and devided into aliquots of 10 mL. From all plates with fluorophore expressing cells,
-                                                one colony was picked and grown in eM9 overnight.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">10<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Transfer to LB cells with plasmids bought from Addgene: pBbS5a and pBbA5c</p>
-                                            <p>Measure OD of overnight cultures in eM9. Dilute them until OD of 0.1 in eM9 supplemented with chloramphenicol.
-                                                Measure kinetic cycle of 7 hours in plate reader.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
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-                                    <div class="row">
-                                        <h2 class="title-style-3">11<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Charlotte & Lara</strong></p>
-                                            <p>Miniprepped plasmids bought from Addgene pBbS5a and pBbA5c from overnight LB cultures; 5 tubes each. The concentrations were confirmed with nanodrop and were between 30 and 60 ng/µL.</p>
-                                        </div>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Restriction of plasmids bought from Addgene pBbS5a and pBbA5c, and PCR products from September 1st with EcoRI and XhoI. The biggest fragment obtained with each plasmid is the backbone, which was gel isolated. Then, backbones and fragments were ligated and transformed into Top10 chemically competent cells.</p>
-                                        </div>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Transformation of</p>
-                                            <ul class="list-unstyled">
-                                                <li>BolA_con pSB1C3</li>
-                                                <li>INP_Sil_Sdom pSB4A5</li>
-                                                <li>OmpA_Sil_Taur pSB4A5</li>
-                                                <li>Sil_Sdom pSB4A5</li>
-                                                <li>Sul_A pSB1C3</li>
-                                                <li>OmpA_Sil_Sdom pSB4A5</li>
-                                                <li>RFP pSB1C3 (positive control)</li>
-                                                <li>RFP pSB4A5 (positive control)</li>
-                                                <li>Sterile MiliQ (negative control)</li>
-                                            </ul>
-                                            <p>in TOP10. Plated on selective agar plates.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">12<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Colony PCR of transformations from yesterday 16 colonies of each transformation were picked, but only analized 15 of each.</p>
-                                            <img src="https://static.igem.org/mediawiki/2016/0/06/T--TU_Delft--Electrophoresis_20160912_1.png" alt="electrophoresis">
-                                            <img src="https://static.igem.org/mediawiki/2016/8/81/T--TU_Delft--Electrophoresis_20160912_2.png" alt="electrophoresis">
-                                            <img src="https://static.igem.org/mediawiki/2016/1/13/T--TU_Delft--Electrophoresis_20160912_3.png" alt="electrophoresis">
-                                            <p>Red arrows point at colonies picked for overnight cultures in selective medium.</p>
-                                        </div>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Measure spectrum of mVenus and mCerulean cells in plate reader. </p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">13<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Plasmid isolation, preparation of samples for sequencing and cryostock of overnight cultures of colonies picked yesterday.</p>
-                                            <p>Sequences of all colonies picked for OmpA_Sil_Taur, INP_Sil_Sdom, Sil_Sdom and SulA were confirmed to be good. In the case of BolA there's a deletion of a single bp, to be fixed by PCR. All sequences for OmpA_Sil_Sdom contained a strange long insert, this silicatein gene version was dropped for further experiments.</p>
-                                        </div>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Put 100µl of TOP10 competent cells into 10ml LB in a falcon tube and incubated overnight at 37 degrees Celsius at 250rpm.</p>
-                                            <p>Made LB and LB agar.</p>
-                                        </div>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Measure spectrum of eM9 cultures in plate reader. </p>
-                                            <p>Sequences of mKate gibson assembled to phaCAB arrived and didn't yield the good results.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">14<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Made ~100 aliquots TOP10 competent cells with culture from yesterday according to protocol. OD600 before putting on ice was 0.34.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">15<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Transformation of plasmids with confirmed sequences from 13<sup>th</sup> of September into BL21. Also, combined with GFP under promoter J23100 in pSB1C3 backbone. All 3 silicatein versions, SulA and the combination of Sil_Sdom and GFP yielded colonies. Other 2 silicateins together with GFP did not yield any positive result.</p>
-                                        </div>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Did positive and negative controls of competent cells made yesterday. Used RFP pSB1C3 for positive control and MiliQ for negative control.</p>
-                                        </div>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Wash 3x in PBS cultures grown in LB expressing fluorophores. Dissolve in PBS and measure spectrum in plate reader.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">16<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Repeat kinetic cycle experiment in plate reader with different settings, because the previous results didn't show good results.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">18<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Transfer from plate to liquid LB colonies with BL21 cells expressing OmpA_Sil_Taur, Sil_Sdom and INP_Sil_Sdom for widefield microscope experiment.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">19<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka & María</strong></p>
-                                            <p>Transfer LB cultures from yesterday to fresh medium and grow until OD reached 0.65. Then added 1mM sterile IPTG to induce.
-                                                Grown in the incubator for another 5 hours. Subsequently added 60µM Sodium Silicate and incubated for 3 hours before putting them in the fridge.</p>
-                                            <p>Diluted and filter sterilized Sodium Silicate in miliQ water up to a concentration of 1M.</p>
-                                            <p>Diluted and filter sterilized Rhodamine 123 in ethanol up to a concentration of 20 mg/mL and added 15µL of this to the indicated cultures.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">20<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Transfer of colonies from 15<sup>th</sup> of September transformations into overnight culture for cryostocking.</p>
-                                            <p><strong>Lycka & Charlotte</strong></p>
-                                            <p>Add 7µM rhodamine to the cultures from yesterday, incubated 10 min. Centrifuged 1.5 mL of culture at 4000 rpm 5 min
-                                                Washed 3x and then resuspended in 400µL PBS. Imaged in widefield microscope with excitation wavelength of 488 nm.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">21<sup>st</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Cryostock of BL21 strains containing sequence confirmed plasmids from yesterday's overnight cultures.</p>
-                                            <p>Transformation into BL21 of combinations of OmpA_Sil_Taur with all fluorophores (separately) and INP_Sil_Sdom with GFP under promoter J23100. <br>
-                                                None of them yielded any colonies. We are going to try reducing the antibiotics concentration in the plates to half because probably the cells are not able to handle the 2 plasmids and such a high concentration of antibiotics.</p>
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Measure OD600 of overnight cultures María prepared. They were around 1.46, 1.35, 1.49 for Sil_Sdom, OmpA_Sil_Taur, INP_Sil_Sdom respectively.
-mL of culture was transfered to 10 mL fresh LB.</p>
-                                            <p>Dissolved and filter sterilized IPTG. 1.2 g in 5 mL.</p>
-                                            <p>Saved cultures in the fridge to continue tomorrow.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">22<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Inoculate 0.25 mL of yesterday's culture in 10 mL fresh LB. Cells did not grow after half a day. Inoculate
-                                                new culture from plate, grown overnight.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">23<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Test silicic acid test, with 1mM aliquot of silic acid.</p>
-                                            <ul class="list-unstyled">
-                                                <li>100 µL 1 mM silicate</li>
-                                                <li>4900 µL miliQ</li>
-                                                <li>3 drops reagent 1</li>
-                                                <li>3 drops reagent 2</li>
-                                                <li>500 µL reagent 3</li>
-                                            </ul>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">24<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Measure OD600 of overnight eM9 cultures, diluted 4 times before measuring.</p>
-                                            <h4>OD600 measurements</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>Genotype</th>
-                                                <th>OD600</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>GFP J23100</td>
-                                                        <td>0.90</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>GFP J23105</td>
-                                                        <td>0.51</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>GFP J23108</td>
-                                                        <td>0.88</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>GFP J23113</td>
-                                                        <td>didnt grow</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>GFP J23117</td>
-                                                        <td>didnt grow</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mVenus</td>
-                                                        <td>0.74</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>mCerulean</td>
-                                                        <td>0.77</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>Dilute until OD 0.1 and divide into aliquots of 100µL in a 96 well plate. Performed kinetic cycle in plate reader.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">26<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Repeat of transformations of 21<sup>st</sup> of September using plates with half the concentration of antibiotics. All of them yielded colonies.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">27<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Transfer of colonies from yesterday's transformations to overnight culture.</p>
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Prepare eM9 an divide into aliquots of 10 mL, inoculate with:</p>
-                                            <ul class="list-unstyled">
-                                                <li>OmpA Sil Taur</li>
-                                                <li>INP Sil Sdom</li>
-                                                <li>Sil Sdom</li>
-                                                <li>BolA</li>
-                                                <li>OmpA Sil Taur</li>
-                                                <li>OmpA Sil Taur + mCerulean</li>
-                                                <li>mCerulean</li>
-                                            </ul>
-                                            <p></p>
-                                            <p>Restreak plates with BL21 OmpA Sil Taur and BolA on fresh LB+CM plates.</p>
-                                            <p>Cryostock OmpA + mCerulean, + mVenus and + all GFPs.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">28<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            <p>Cryostock of BL21 cells from overnight culture from yesterday.</p>
-                                            <p>Fixing of BolA mutation by PCR.</p>
-                                            <img src="https://static.igem.org/mediawiki/2016/d/d4/T--TU_Delft--Electrophoresis_20160928.png" alt="electrophoresis">
-                                            <p>Picture on the left shows simulated agarose gel. Picture on the right shows the actual agarose gel. PCR didn't work, we are going to try again with a gradient of annealing temperatures to see if this is the cause of the problem.</p>
-                                            <p><strong>Lycka</strong></p>
-                                            <p>Measure OD600 of overnight eM9 cultures, diluted 4 times before measuring.</p>
-                                            <h4>OD600 measurements</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>Genotype</th>
-                                                <th>OD600</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>INP Sil Sdom</td>
-                                                        <td>1.15</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>BolA</td>
-                                                        <td>1.18</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>OmpA Sil Taur</td>
-                                                        <td>1.26</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Sil Sdom</td>
-                                                        <td>1.00</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>Dilute until OD 0.05 in 200 mL eM9 and grown in shaker at 37 degrees. Cultures didn't grow after half a day.</p>
-                                            <p></p>
-                                            <p>Prepared calibration curve for silicate test. Dilutions made with 1 mM silic acid stock.</p>
-                                            <h4>Calibration curve</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>concentration (µM)</th>
-                                                <th>silica stock (µL)</th>
-                                                <th>miliQ (µL)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>100</td>
-                                                        <td>500</td>
-                                                        <td>4500</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>80</td>
-                                                        <td>400</td>
-                                                        <td>4600</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>60</td>
-                                                        <td>300</td>
-                                                        <td>4700</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>40</td>
-                                                        <td>200</td>
-                                                        <td>4800</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>20</td>
-                                                        <td>100</td>
-                                                        <td>4900</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>10</td>
-                                                        <td>50</td>
-                                                        <td>4950</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>5</td>
-                                                        <td>25</td>
-                                                        <td>4975</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>0</td>
-                                                        <td>0</td>
-                                                        <td>5000</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p></p>
-                                            <p>Prepare overnight eM9 cultures for:</p>
-                                            <ul class="list-unstyled">
-                                                <li>Silic acid test: OmpA_Sil_Taur, INP_Sil_Sdom, Sil_Sdom, BolA</li>
-                                                <li>Laser setup: mCerulean, OmpA_Sil_Taur, mCerulean + OmpA_Sil_Taur</li>
-                                                <li>TEM: OmpA_Sil_Taur, INP_Sil_Sdom, Sil_Sdom</li>
-                                            </ul>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">29<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>Tessa</strong></p>
-                                            <p>Made ~50 aliquots BL21 competent cells according to protocol. OD600 before putting on ice was 0.30.</p>
-                                            <p><strong>María</strong></p>
-                                            <p>PCR to fix BolA mutation.</p>
-                                            <img src="https://static.igem.org/mediawiki/2016/b/bb/T--TU_Delft--Electrophoresis_20160929.png" alt="electrophoresis">
-                                            <p>All annealing temperatures resulted in a product of the desired length.<br>
-. 66ºC; 2. 65ºC; 3. 63.4ºC; 4. 60ºC</p>
-                                            <p><strong>Lycka & María</strong></p>
-                                            <p>Measure OD600 of overnight eM9 cultures, diluted 4 times before measuring.</p>
-                                            <h4>OD600 measurements</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>Genotype</th>
-                                                <th>OD600</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>INP Sil Sdom</td>
-                                                        <td>0.77</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>BolA</td>
-                                                        <td>0.74</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>OmpA Sil Taur</td>
-                                                        <td>0.75</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Sil Sdom</td>
-                                                        <td>0.68</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>Inoculate 4 µL in 150 µL fresh eM9. After induction Erlenmeyer fell over and content was not usefull anymore.</p>
-                                            <p></p>
-                                            <p>Measure OD600 of overnight LB cultures, diluted 4 times before measuring.</p>
-                                            <h4>OD600 measurements</h4>
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>Genotype</th>
-                                                <th>OD600</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>INP Sil Sdom</td>
-                                                        <td>0.73</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>OmpA Sil Taur</td>
-                                                        <td>0.76</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Sil Sdom</td>
-                                                        <td>0.91</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p>Inoculate 100 µL in 10 mL fresh medium. Grow until OD 0.55 and add 10 µL 1 M IPTG.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">30<sup>th</sup> September 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong>María</strong></p>
-                                            Silicic acid test: since cells grew too slow in eM9 medium we checked if LB absorbance spectrum interfered with the absorbance of the final product of the test (~815nm). It didn't so we decided to grow the cells in LB medium.<br>
-                                            Measured OD600 of overnight cultures with a 1/4 dilution:
-                                            <table class="notebook table table-style-1">
-                                                <thead>
-                                                <th>Genotype</th>
-                                                <th>OD600</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>OmpA Sil Taur</td>
-                                                        <td>0.77</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>INP Sil Sdom</td>
-                                                        <td>0.74</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Sil Sdom</td>
-                                                        <td>0.75</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>BolA</td>
-                                                        <td>0.68</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <p><strong> Carmen & Charlotte </strong></p>
-                                            <p>Tested whether adding silicatein cells to a solar panel had any effect. We tested the strain expressing OmpA-fused silicatein which was incubated with silica, so it had a silicate coating.  The cells were washed 3x in PBS. We connected a multimeter that was measuring voltage to a solar cell with a limited area exposed to light. We measured the output power of the solar cell at the same time as the light intensity of the lamp. We repeated this experiment with the same area of the solar cell covered in PBS and covered in PBS + silicatein-expressing cells.  </p>
-                                            <p><strong> Charlotte & Liza </strong></p>
-                                            <p>Tested the mCerulean-expressing cells in our self-built hardware setup. The cells were grown in LB and washed 3x with PBS. The cells were fixed on a slide by pipetting them under 3% agarose-TAE pads.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <a href="#notes" class="btn btn-info" role="button" onclick="$('html,body').animate({scrollTop: $('#notes').offset().top}, 'slow');" style="text-decoration:none; color:#f3f4f4; float:right;">Back to Top</a>
-                                </div>
-                            </div>
-                        </div>
-                        <div class="panel panel-default">
-                            <a class="collapsed" data-toggle="collapse" data-parent="#accordion" href="#collapseFive" aria-expanded="false" aria-controls="collapseFive">
-                                <div class="panel-heading" role="tab" id="headingFive">
-                                    <h4 class="panel-title">
-                                        October
-                                    </h4>
-                                </div>
-                            </a>
-                            <div id="collapseFive" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingFive">
-                                <div class="panel-body">
-                                    <!-- put an activity here -->
-                                    <div class="row">
-                                        <h2 class="title-style-3">4<sup>th</sup> October 2016</h2>
-                                        <div class="col-md-12">
-                                            <p><strong> Charlotte & Liza </strong></p>
-                                            <p>Tested the mCerulean-expressing cells, the OmpA-silicatein expressing cells and OmpA-silicatein + mCerulean expressing cells in our self-built hardware setup. The cells were grown in LB, spun down and resuspended in PBS. The cells were fixed on a slide by pipetting them under 3% agarose-TAE pads. The cells were excited at 405 nm and imaged at different excitation energies.</p>
-                                        </div>
-                                    </div>
-                                    <!-- end activity-->
-                                    <a href="#notes" class="btn btn-info" role="button" onclick="$('html,body').animate({scrollTop: $('#notes').offset().top}, 'slow');" style="text-decoration:none; color:#f3f4f4; float:right;">Back to Top</a>
-                                </div>
-                            </div>
-                        </div>
-                    </div>
-                </div>
-                <div class="protocols">
-                    <span class="anchor" id="protocols"></span>
-                    <h2 class="title-style-1">Protocols<span class="title-under"></span></h2>
-                    <h3>During our lab work we used the following protocols.</h3>
-                    <div class="container protocols">
-                        <div role="tabpanel">
-                            <!-- Nav tabs -->
-                            <ul class="notebook nav nav-tabs" role="tablist">
-                                <li role="presentation" class="active"><a href="#competentcells" aria-controls="home" role="tab" data-toggle="tab">Chemically competent Cells</a></li>
-                                <li role="presentation"><a href="#pcr" aria-controls="settings" role="tab" data-toggle="tab">PCR (Phusion)</a></li>
-                                <li role="presentation"><a href="#gelisolation" aria-controls="settings" role="tab" data-toggle="tab">Gel Isolation (From Promega Wizard™ Kit)</a></li>
-                                <li role="presentation"><a href="#restriction" aria-controls="settings" role="tab" data-toggle="tab">Restriction (NEB enzymes)</a></li>
-                                <li role="presentation"><a href="#ligation" aria-controls="settings" role="tab" data-toggle="tab">Ligation</a></li>
-                                <li role="presentation"><a href="#transformation" aria-controls="profile" role="tab" data-toggle="tab">Transformation (TOP10)</a></li>
-                                <li role="presentation"><a href="#bl21" aria-controls="messages" role="tab" data-toggle="tab">Transformation (BL21)</a></li>
-                                <li role="presentation"><a href="#oneshot" aria-controls="messages" role="tab" data-toggle="tab">Transformation (OneShot<sup>®</sup> TOP10)</a></li>
-                                <li role="presentation"><a href="#NEB" aria-controls="settings" role="tab" data-toggle="tab">Colony PCR (Q5<sup>®</sup> High-Fidelity 2X Master Mix)</a></li>
-                                <li role="presentation"><a href="#gotaq" aria-controls="settings" role="tab" data-toggle="tab">Colony PCR (GoTaq)</a></li>
-                                <li role="presentation"><a href="#dreamtaq" aria-controls="settings" role="tab" data-toggle="tab">Colony PCR (DreamTaq)</a></li>
-                                <li role="presentation"><a href="#electrophoresis" aria-controls="settings" role="tab" data-toggle="tab">DNA Electrophoresis</a></li>
-                                <li role="presentation"><a href="#cultures" aria-controls="settings" role="tab" data-toggle="tab">Overnight Cell Cultures</a></li>
-                                <li role="presentation"><a href="#cryostock" aria-controls="settings" role="tab" data-toggle="tab">Cell Frozen Stock</a></li>
-                                <li role="presentation"><a href="#miniprep" aria-controls="settings" role="tab" data-toggle="tab">Plasmid Isolation (Promega PureYield™ Plasmid Miniprep Kit)</a></li>
-                                <li role="presentation"><a href="#purification" aria-controls="settings" role="tab" data-toggle="tab">Restriction Product Purification (from Promega Wizard™ Kit)</a></li>
-                                <li role="presentation"><a href="#polysilicate" aria-controls="settings" role="tab" data-toggle="tab">Polysilicate layer in silicatein expressing cells</a></li>
-                                <li role="presentation"><a href="#growth" aria-controls="settings" role="tab" data-toggle="tab">Growth study</a></li>
-                                <li role="presentation"><a href="#plate" aria-controls="settings" role="tab" data-toggle="tab">Plate reader</a></li>
-                                <li role="presentation"><a href="#Test" aria-controls="settings" role="tab" data-toggle="tab">Silicic acid test</a></li>
-                            </ul>
-                            <!-- Tab panes -->
-                            <div class="tab-content">
-                                <div role="tabpanel" class="tab-pane active" id="competentcells">
-                                    <h2>Chemically competent cells</h2>
-                                    <ol>
-                                        <li>Grow overnight culture from single colony or competent cells aliquot.</li>
-                                        <li>Inoculate 1:100 in the desired volume of LB.</li>
-                                        <li>Grow to OD 600 ~0.35 (important that it is close to 0.35, 0.3-0.4 is good).</li>
-                                        <li>Chill on ice for 20 min (it can chill for a 1-2 hours).</li>
-                                        <li>Centrifuge at 8000g for 8 minutes in Falcon tubes, decant supernatant.</li>
-                                        <li>Resuspend by pipetting gently in 1/10 (of the volume of LB from step 2) of ice-cold TSS.<br>
-                                            <u>NOTE</u>: It can take a while to break up the pellet (you can use a 25 mL pipette and scrape the pellet off the side of the tube before pipetting).</li>
-                                        Recipe for TSS (100 mL):
-                                        <ul>
-                                            <li>10 g PEG 3350</li>
-                                            <li>5 mL DMSO</li>
-                                            <li>2 mL 1 M MgCl<sub>2</sub></li>
-                                            <li>LB to 100 mL</li>
-                                        </ul>
-                                        Filter sterilize and chill to 4 degrees Celsius
-                                        <li>Freeze 100 µL aliquots in liquid nitrogen & store at -80 degrees C in 1,5mL Eppendorf tubes.</li>
-                                        <li>ALWAYS do a transformation positive control using a plasmid that you know is efficiently taken up by competent cells to confirm competency.</li>
-                                        <li>Just as important: ALWAYS do a negative control transformation (add untransformed cells to a plate with antibiotics) to confirm that you don't have an antibiotic-resistant contaminant strain in your competent cell stock. This happens from time to time and can cause lots of agony!</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="pcr">
-                                    <h2>PCR (Phusion polymerase)</h2>
-                                    <ol>
-                                        <li>Prepare the mix for all the samples in a single 1.5mL tube (mind pipetting error!). For one sample:
-                                            <table class="protocols table table-style-1">
-                                                <thead>
-                                                <th>Component</th>
-                                                <th>Volume (µL) for 20µL reaction</th>
-                                                <th>Volume (µL) for 50µL reaction</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>5X Phusion HF Buffer</td>
-                                                        <td>4</td>
-                                                        <td>10</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>10mM dNTPs</td>
-                                                        <td>0.4</td>
-                                                        <td>1</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>FW Primer</td>
-                                                        <td>1</td>
-                                                        <td>2.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>RV Primer</td>
-                                                        <td>1</td>
-                                                        <td>2.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Phusion polymerase</td>
-                                                        <td>0.2</td>
-                                                        <td>0.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Template DNA (<250ng)</td>
-                                                        <td>Variable</td>
-                                                        <td>Variable</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Nuclease-free H<sub>2</sub>O</td>
-                                                        <td>Up to 20µL</td>
-                                                        <td>Up to 50µL</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                            <u>NOTE</u>: The 250ng of template DNA are approximate, choose a volume that works fine for all your samples and fill it in the excel sheet so you are able to prepare a mix for all samples at the same time.</li>
-                                        <li>Add the total volume per sample (without DNA) to each PCR tube.</li>
-                                        <li>Add the template DNA to each tube.</li>
-                                        <li>Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):
-                                            <table class="protocols table table-style-1">
-                                                <thead>
-                                                <th>Step</th>
-                                                <th>Temperature (ºC)</th>
-                                                <th>Time</th>
-                                                <th>  </th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>Initial denaturation</td>
-                                                        <td>98</td>
-                                                        <td>30sec</td>
-                                                        <td>  </td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Denaturation</td>
-                                                        <td>98</td>
-                                                        <td>10sec</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Annealing</td>
-                                                        <td>Tm-3</td>
-                                                        <td>20sec</td>
-                                                        <td rowspan="2">x30 cycles</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Extension</td>
-                                                        <td>72</td>
-                                                        <td>15-30sec/1kb</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Final extension</td>
-                                                        <td>72</td>
-                                                        <td>5min</td>
-                                                        <td>  </td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td></td>
-                                                        <td>4</td>
-                                                        <td>Hold</td>
-                                                        <td>  </td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                        </li>
-                                        <li>Run a DNA electrophoresis following the <i>DNA electrophoresis</i> protocol.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="gelisolation">
-                                    <h2>Gel isolation</h2>
-                                    <ol>
-                                        <li>Run a DNA electrophoresis (following the <i>DNA electrophoresis</i> protocol), loading the full volume of PCR or restriction product into the agarose gel. If necessary, use multiple wells.</li>
-                                        <li>Cut the band (or bands) corresponding to the fragment of interest with a razor blade and put it in a pre-weighted 1.5mL tube, then calculate the weight of the gel fragment.</li>
-                                        <li>Add 10µL of Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50-65ºC until gel slice is completely dissolved.</li>
-                                        <li>Pre-warm an aliquot of Nuclease-free water at 50-65ºC.</li>
-                                        <li>Insert SV Minicolumn into Collection Tube and label both of them according to the labelling of your samples.</li>
-                                        <li>Transfer the dissolved gel mixture to the Minicolumn assembly. Incubate at room temperature for 1 minute. <br>
-                                            <u>NOTE</u>: When pipetting into the column, aim the pipette to the wall not the membrane to avoid damaging it.</li>
-                                        <li>Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.</li>
-                                        <li>Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.</li>
-                                        <li>Add 700µL of Membrane Wash Solution (if it is the first use, dilute it with 95% ethanol following the bottle’s instructions).</li>
-                                        <li>Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.</li>
-                                        <li>Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.</li>
-                                        <li>Repeat steps 9-11 with 500µL of Membrane Wash Solution and centrifuging for 5 minutes.</li>
-                                        <li>Once the Collection Tube is empty, centrifuge the Minicolumn assembly at maximum speed for 1 minute with the microcentrifuge lid open to allow ethanol full evaporation.</li>
-                                        <li>Transfer the SV Minicolumn to an empty 1.5mL tube carefully labelled.</li>
-                                        <li>Add 500µL of the pre-warmed water (300µL for higher concentrations or when small amounts of DNA are suspected) directly to the centre of the SV Minicolumn, without touching the membrane with the pipette tip.</li>
-                                        <li>Incubate at room temperature for 5 minutes.</li>
-                                        <li>Centrifuge at maximum speed for 1 minute.</li>
-                                        <li>Discard the SV Minicolumn, cap the tube containing the eluted DNA and keep at 4ºC (for immediate use) or -20ºC (for storage).</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="restriction">
-                                    <h2>Restriction</h2>
-                                    <ol>
-                                        <li>Get the detailed protocol for restriction from the following <a href="http://nebcloner.neb.com/#!/redigest">website</a>, according to the enzymes used. When high restriction product concentrations are needed (generally for backbones) the amount of DNA can be increased to 3-5g. The volume of DNA necessary can be obtained from the concentration measured at NANODROP.</li>
-                                        <li>Mix all the components stated by this restriction tool in a 1.5mL Eppendorf tube and incubate at 37ºC for 1-2h.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="ligation">
-                                    <h2>Ligation</h2>
-                                    <ol>
-                                        </body>                                    <li>Make your own ligation protocol.</li>
-                                        <form id="form1">
-                                            <title>Ligation Protocol</title>
-                                            <body>
-                                                <h2>Ligation Protocol</h2>
-                                                <fieldset>
-                                                    <label>Vector length (bp) :</label> <input type="text" name="VL" value="" /><br /><br />
-                                                    <label>Vector mass (ng)   :</label>  <input type="text" name="VM" value="" / /><br /><br />
-                                                    <label>Vector concentration (ng/µL) :</label> <input type="text" name="VC" value="" //><br /><br />
-                                                    <label>Insert length (bp)  :</label>  <input type="text" name="IL"value="" / /><br /><br />
-                                                    <label>Insert concentration (ng/µL) :</label> <input type="text" name="IC" value="" //><br /><br />
-                                                    <label>Ratio Insert/Vector  :</label>  <input type="text" name="R" value="3" //><br /><br />
-                                                </fieldset>
-                                        </form>
-                                        <button onclick="myFunction()">Calculate</button>
-                                        <p id="Tool"></p>
-                                        <script>
-                                            function myFunction() {
-                                                var x, vl, vm, vc, il, ic, r, T;
-                                                x = document.getElementById("form1");
-                                                vl = x.elements["VL"].value;
-                                                vm = x.elements["VM"].value;
-                                                vc = x.elements["VC"].value;
-                                                il = x.elements["IL"].value;
-                                                ic = x.elements["IC"].value;
-                                                r = x.elements["R"].value;
-                                                T = Number(vl) + Number(vm);
-                                                document.getElementById("Tool").innerHTML = "";
-                                                if (isNaN(vl) || isNaN(vm) || isNaN(vc) || isNaN(il) || isNaN(ic) || isNaN(r)) {
-                                                    document.getElementById("Tool").innerHTML += " Value is not valid! Please insert a number <br>";
-                                                } else if (vl <= 0 || vm <= 0 || vc <= 0 || il <= 0 || ic <= 0 || r <= 0) {
-                                                    document.getElementById("Tool").innerHTML += " Values have to be larger than zero. Please insert a valid 		 number <br>";
-                                                } else {
-                                                    vv = Number(vm) / Number(vc);
-                                                    im = Number(vm) / Number(vl) * Number(r) * Number(il);
-                                                    iv = im / Number(ic);
-                                                    W = 20 - iv - vv - 2 - 1;
-                                                    ;
-                                                    document.getElementById("Tool").innerHTML += "For a total volume of 20 µL please add: <br><br> "
-                                                    document.getElementById("Tool").innerHTML += "- " + vv.toFixed(1) + " µL Vector<br>"
-                                                    document.getElementById("Tool").innerHTML += "- " + iv.toFixed(1) + " µL Insert<br>"
-                                                    document.getElementById("Tool").innerHTML += "- 1.0 µL Ligase <br>"
-                                                    document.getElementById("Tool").innerHTML += "- 2.0 µL Buffer <br>"
-                                                    document.getElementById("Tool").innerHTML += "- " + W.toFixed(1) + " µL Nuclease-Free water<br>"
-                                                }
-                                            }
-                                        </script>
-                                        </body>
-                                        <body>
-                                        <li>Mix all the components in a tube (always add the T4 ligase at the end).</li>
-                                        <li>Incubate for at least 3 hours at 16ºC.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="transformation">
-                                    <h2>Transformation (Top10 chemically competent cells)</h2>
-                                    <ol>
-                                        <li>Get as many aliquots of competent cells (100L) from the -80ºC freezer as transformations to be done and put them on ice for 10-15min.<br>
-                                            <u>NOTE</u>: Don’t forget positive (RFP in the backbone you are using, check plasmids box in the -20ºC freezer) and negative controls (no DNA).</li>
-                                        <li>Add to the 100µL of competent cells:
-                                            <ol type="i">
-                                                <li>1µL of plasmid or 2µL of ligation product.</li>
-                                                <li>20µL 5X KCM buffer.</li>
-                                                <li>Sterile water up to 200µL.</li>
-                                            </ol>
-                                        </li>
-                                        <li>Mix by flicking the tube.</li>
-                                        <li>Incubate on ice for 20-30 minutes.</li>
-                                        <li>Heat shock at 42ºC for exactly 90 seconds.</li>
-                                        <li>Return the cells to ice for 1-2 minutes.</li>
-                                        <li>Add 400µL of fresh LB.</li>
-                                        <li>Incubate at 37ºC with shaking for 1 hour.<br>
-                                            <u>NOTE</u>: Not at the thermoblock, use a shaking incubator.</li>
-                                        <li>To increase the concentration of cells before plating, centrifuge the tubes at 4000rpm for 3 minutes, remove 500L of medium, resuspend the pellet in the remaining volume.</li>
-                                        <li>Plate the rest of the volume of the transformation on selective LB plates and incubate at 37ºC overnight.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="bl21">
-                                    <h2>Transformation (BL21 competent cells)</h2>
-                                    <ol>
-                                        <li>Put a tube of BL21 Competent E. coli cells on ice for 10 minutes.</li>
-                                        <li>Add 2µL of plasmid DNA to the cell mixture.</li>
-                                        <li>Mix by flicking the tube.</li>
-                                        <li>Place the tube on ice for 30 minutes.</li>
-                                        <li>Heat shock at 42ºC for exactly 10 seconds.</li>
-                                        <li>Place on ice for 5 minutes.</li>
-                                        <li>Add 950µL of SOC medium at room temperature.</li>
-                                        <li>Incubate at 37ºC with shaking for 1 hour.<br>
-                                            <u>NOTE</u>: Not at the thermoblock, use a shacking incubator.</li>
-                                        <li>To increase the concentration of cells before plating, centrifuge the tubes at 4000rpm for 3 minutes, remove 700µL of medium, resuspend the pellet in the remaining volume.</li>
-                                        <li>Plate 70µL of the transformation on selective LB plates and incubate at 37ºC overnight.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="oneshot">
-                                    <h2>Transformation (OneShot<sup>&reg;</sup> Top10 Chemically competent cells)</h2>
-                                    <ol>
-                                        <li>Get half the amount of tubes of OneShot<sup>&reg;</sup> TOP10 chemically competent cells (tubes with a purple lid) than the transformations to be done from the -80ºC freezer and put them on ice for 10-15min.</li>
-                                        <li>Divide the volume of competent cells of each tube in 2. Thus, transfer 25L to a new sterile tube.</li>
-                                        <li>Add 1µL of plasmid or 2µL of ligation product into each tube and mix by flicking.</li>
-                                        <li>Incubate on ice for 30 min.</li>
-                                        <li>Heat shock the cells for 30 seconds at 42ºC.</li>
-                                        <li>Put back on ice for 2 minutes.</li>
-                                        <li>Add 250µL of LB medium.</li>
-                                        <li>Incubate at 37ºC with shaking for 1 hour.<br>
-                                            <u>NOTE</u>: Not at the thermoblock, use a shacking incubator.</li></li>
-                                        <li>To increase the concentration of cells before plating, centrifuge the tubes at 4000rpm for 3 minutes, remove 200µL of medium, resuspend the pellet in the remaining volume.</li>
-                                        <li>Plate the rest of the volume of the transformation on selective LB plates and incubate at 37ºC overnight.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="NEB">
-                                    <h2>Colony PCR (Q5<sup>&reg;</sup> High-Fidelity 2X Master Mix)</h2>
-                                    <ol>
-                                        <li>Choose the colonies to be picked for testing and number them. When available pick at least 8 colonies per plate, or more if there are slots left in the PCR machine (it fits 25 samples).<br>
-                                            <u>NOTE</u>: It is more convenient if the numbering is continued between plates of the same batch instead of starting at 1 in each plate. This way there is only one set of numbers per date.<br>
-                                            <u>NOTE</u>: it is possible that not all samples fit in one PCR and then more than one would need to be performed, so keep that in mind for the timing of experiments. But don’t be sad, that would mean that the transformation worked so well that you have a lot of colonies to choose from!! &#9786;</li>
-                                        <li>Prepare the mix for all the samples in a single 1.5mL tube (mind pipetting error!). For one sample:
-                                            <table class="protocols table table-style-1">
-                                                <thead>
-                                                <th>Component</th>
-                                                <th>Volume (µL)</th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>Q5<sup>&reg;</sup> HF 2X Master Mix</td>
-                                                        <td>12.5</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Primer VF2</td>
-                                                        <td>1.25</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Primer VR</td>
-                                                        <td>1.25</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Nuclease-free H<sub>2</sub>O</td>
-                                                        <td>10</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Total</td>
-                                                        <td>25</td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                        </li>
-                                        <li>Pipette 25µL of mix into each PCR tube.</li>
-                                        <li>Under sterile conditions, pick a colony making sure that part of it stays on the plate for later use and soak it in the content of the PCR tube. When finished, put them back in the incubator so they grow for a bit longer while the PCR runs.</li>
-                                        <li>Put the tubes in the PCR machine and apply the following program (applicable for the primers mentioned above, for other sets of primers the annealing temperature needs to be adjusted):
-                                            <table class="protocols table table-style-1">
-                                                <thead>
-                                                <th>Step</th>
-                                                <th>Temperature (ºC)</th>
-                                                <th>Time</th>
-                                                <th>  </th>
-                                                </thead>
-                                                <tbody>
-                                                    <tr>
-                                                        <td>Cell lysis and initial denaturation</td>
-                                                        <td>95</td>
-                                                        <td>15min</td>
-                                                        <td>  </td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Denaturation</td>
-                                                        <td>95</td>
-                                                        <td>30sec</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Annealing</td>
-                                                        <td>53</td>
-                                                        <td>30sec</td>
-                                                        <td rowspan="2">x30 cycles</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Extension</td>
-                                                        <td>72</td>
-                                                        <td>30sec/1kb</td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td>Final extension</td>
-                                                        <td>72</td>
-                                                        <td>5min</td>
-                                                        <td>  </td>
-                                                    </tr>
-                                                    <tr>
-                                                        <td></td>
-                                                        <td>4</td>
-                                                        <td>Hold</td>
-                                                        <td>  </td>
-                                                    </tr>
-                                                </tbody>
-                                            </table>
-                                        </li>
-                                        <li>While the PCR is running, prepare a 0.5mL tube with 2µL loading buffer for each PCR sample and one tube with 4µL of 1Kb DNA ladder (or 8µL if 2 gels are needed). This way you can take these tubes into the EtBr area and don’t worry about how to handle the PCR tubes.</li>
-                                        <li>Cast an agarose gel following the <i>DNA electrophoresis</i> protocol.</li>
-                                        <li>Prepare an agarose gel with SNAPGENE for comparison with the actual results.</li>
-                                        <li>Once the PCR program is finished, transfer 8L of PCR product into the tubes containing the loading buffer and follow the <i>DNA electrophoresis</i> protocol.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="gotaq">
-                                    Lorem ipsum dolor sit amet, consectetur adipisicing elit. In consequatur mollitia, libero quisquam impedit obcaecati, dignissimos, eum similique minima ab amet eos sequi distinctio qui modi? Possimus quos, fugit quia.</div>
-                                <div role="tabpanel" class="tab-pane" id="dreamtaq">
-                                    Lorem ipsum dolor sit amet, consectetur adipisicing elit. Molestiae aut culpa a commodi. Quidem asperiores aliquid sequi incidunt quas soluta eum ab fugiat deleniti in at iste, illum ipsam nisi.</div>
-                                <div role="tabpanel" class="tab-pane" id="electrophoresis">
-                                    <h2>DNA electrophoresis</h2>
-                                    <p><b>Work clean!</b> Handle with gloves all material labelled as EtBr contaminated, don’t take it outside of the EtBr area and don’t touch with gloves anything that is not labelled as EtBr contaminated.</p>
-                                    <ol>
-                                        <li>Prepare TAE buffer: take the 10X concentrated TAE from the chemicals cabinet and dilute it 10 times with demi water. For 500mL, add 50mL to 450mL of dH2O.</li>
-                                        <li>Weigh agarose for a 1% gel. For 200mL, 2g of agarose is necessary.</li>
-                                        <li>Mix the TAE solution with the agarose and heat the solution until it is completely dissolved.</li>
-                                        <li>Add Ethidium bromide to the gel mould. 2.5L for the small gel and 5L for the big one.</li>
-                                        <li>Pour the solution into the mould making sure there are no bubbles and that the EtBr is completely mixed. Let it solidify (approx. 20 minutes).</li>
-                                        <li>Transfer the gel to the electrophoresis cell minding the arrow that indicates the direction of DNA migration, remove the combs and cover it with TAE.</li>
-                                        <li>Prepare tubes with 5/6 of PCR product and 1/6 of loading buffer.</li>
-                                        <li>Load the molecular weight marker (ladder) in the first well (check the appropriate volume for each marker, generally 5L works fine) and load the samples in the other wells, according to the order in your lab journal.<br>
-                                            <u>NOTE</u>: Do not contaminate the loading buffer and ladder with EtBr! Do not touch it while wearing a glove.</li>
-                                        <li>Connect the cables following the colour code and run at 100-130V for 30-60 min.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="purification">
-                                    <h2>Restriction product purification</h2>
-                                    <ol>
-                                        <li>For restriction products containing more than one large fragment follow the first 3 steps of the <i>Gel isolation</i> protocol.<br>
-                                            When restriction yields only one large fragment, add an equal volume of Membrane Binding Solution to the volume of restriction product.</li>
-                                        <li>Pre-warm an aliquot of Nuclease-free water at 50-65ºC.</li>
-                                        <li>Insert SV Minicolumn into Collection Tube and label both of them according to the labelling of your samples.</li>
-                                        <li>Transfer the dissolved gel mixture to the Minicolumn assembly. Incubate at room temperature for 1 minute. <br>
-                                            <u>NOTE</u>: When pipetting into the column, aim the pipette to the wall not the membrane to avoid damaging it.</li>
-                                        <li>Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.</li>
-                                        <li>Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.</li>
-                                        <li>Add 700µL of Membrane Wash Solution (if it is the first use, dilute it with 95% ethanol following the bottle’s instructions).</li>
-                                        <li>Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.</li>
-                                        <li>Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.</li>
-                                        <li>Repeat steps 9-11 with 500µL of Membrane Wash Solution and centrifuging for 5 minutes.</li>
-                                        <li>Once the Collection Tube is empty, centrifuge the Minicolumn assembly at maximum speed for 1 minute with the microcentrifuge lid open to allow ethanol full evaporation.</li>
-                                        <li>Transfer the SV Minicolumn to an empty 1.5mL tube carefully labelled.</li>
-                                        <li>Add 500µL of the pre-warmed water (300µL for higher concentrations or when small amounts of DNA are suspected) directly to the centre of the SV Minicolumn, without touching the membrane with the pipette tip.</li>
-                                        <li>Incubate at room temperature for 5 minutes.</li>
-                                        <li>Centrifuge at maximum speed for 1 minute.</li>
-                                        <li>Discard the SV Minicolumn, cap the tube containing the eluted DNA and keep at 4ºC (for immediate use) or -20ºC (for storage).</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="cryostock">
-                                    <h2>Cell frozen stock</h2>
-                                    <b>Work sterile!</b> If plasmid DNA needs to be purified from the overnight culture, remove the necessary volume from the Falcon tube before the cryostock procedure.
-                                    <ol>
-                                        <li>Centrifuge the 50mL tubes at 2000rpm for 10 min.</li>
-                                        <li>Decant the supernatant without disturbing the pellet.</li>
-                                        <li>Add fresh LB medium, around 1/3 of the starting volume of the culture.</li>
-                                        <li>Mix by vortexing until the pellet is completely resuspended.</li>
-                                        <li>Add sterile 80% glycerol solution, the same volume as fresh LB in step 4.</li>
-                                        <li>Mix by vortexing.</li>
-                                        <li>Make 1.5mL aliquots in cryotubes and label them with the cell type, the BioBrick and backbone they contain (if applicable) and the date.</li>
-                                        <li>Store them at -80ºC and update the inventory in the drive folder.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="miniprep">
-                                    <h2>Plasmid isolation</h2>
-                                    <ol>
-                                        <li>Pre-warm an aliquot of Nuclease-free water at 50-60ºC.</li>
-                                        <li>Add 1.5 mL of bacterial culture in LB medium to a 1.5 mL micro-centrifuge tube. Centrifuge that tube at max speed for 3 min.<br>
-                                            <u>NOTE</u>: For higher concentrations repeat step 1 in the same tube.<br>
-                                            <u>NOTE</u>: If cryostocks need to be prepared from the same overnight culture tube, remove the necessary volume for plasmid isolation under sterile conditions!</li>
-                                        <li>Remove the supernatant.</li>
-                                        <li>Resuspend the pellet in 600 µL of MiliQ water.</li>
-                                        <li>Add 100 µL of Cell Lysis Buffer, and mix by inverting 6 times. The color change to blue indicates complete lysis.</li>
-                                        <li>Add 350 µL of cold (4-8ºC) Neutralization Buffer, and mix by inverting the tube. The color change to yellow indicates total neutralization.</li>
-                                        <li>Introduce a PureYield Minicolumn into a PureYield Collection tube.</li>
-                                        <li>Centrifuge at maximum speed for 3 minutes, and transfer the supernatant to the Minicolumn assembly.<br>
-                                            <u>NOTE</u>: When pipetting into the column, aim the pipette to the wall not the membrane to avoid damaging it.</li>
-                                        <li>Centrifuge at maximum speed for 15 seconds and discard the flowthrough.</li>
-                                        <li>Add 200 µL of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed for 15 seconds. It is not necessary to empty the Collection Tube now.</li>
-                                        <li>Add 400 µL of Column Wash Solution to the minicolumn, and centrifuge at maximum speed for 30 seconds.</li>
-                                        <li>Transfer the PureYield Minicolumn to a clean 1.5 mL tube, add 30 µL (or 50L if you expect high concentrations) of pre-warmed MiliQ water directly to the minicolumn matrix. Let stand for 5 minutes at room temperature.</li>
-                                        <li>Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute plasmidic DNA. Cap the tube, and store the DNA solution at -20 ºC (or keep it at 4ºC if it is to be used immediately).<br>
-                                            <u>NOTE</u>: If possible, put the lids in line with the circle of the centrifuge (so not pointing out or inwards) so that the caps don’t break while centrifuging.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="cultures">
-                                    <h2>Overnight cell cultures</h2>
-                                    <ol>
-                                        <li>Prepare 20mL of selective LB (with the antibiotic your cells are resistant to) per colony to be picked.<br>
-                                            <u>NOTE</u>: You can use a sterile bottle to prepare the total volume.</li>
-                                        <li>Transfer 10mL of selective LB to a 50mL Falcon tube. Prepare 2 tubes per colony: 1 for miniprep and 1 for cryostocks.</li>
-                                        <li>Pick 1 colony using an inoculation loop and soak it in the LB. Make sure you have cells left for the second tube.</li>
-                                        <li>Take the tubes to room F1.690 and incubate overnight at 37ºC in the shaking incubator.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="polysilicate">
-                                    <h2>Polysilicate layer in silicatein expressing cells</h2>
-                                    <ol>
-                                        <li>Inoculate a fresh culture of cells from an overnight culture. Make a dilution of around 1/100 of the overnight culture into fresh selective LB.</li>
-                                        <li>Incubate in the shaking incubator at 37ºC and 250 rpm until it reaches exponential phase (just measure OD600 from time to time until duplication time is close to 20 min).</li>
-                                        <li>Induce the culture with 1/1000 volume of IPTG 1M.</li>
-                                        <li>Incubate in the shaking incubator at 37ºC and 250 rpm for 3h.</li>
-                                        <li>Add sodium silicate to a final concentration of 60µM from the 1mM stock (maybe new stock needs to be made from the 1M stock, keep sterile!!).</li>
-                                        <li>Incubate at 37ºC and 250 rpm for 3-4h, after this the cells are ready for further experiments.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="growth">
-                                    <h2>Growth study</h2>
-                                    <ol>
-                                        <li>Follow the <i>Polysilicate layer</i> protocol until step 5.</li>
-                                        <li>Take a 100µL sample directly after adding the sodium silicate, this is t=0.</li>
-                                        <li>Incubate at 37ºC and 250 rpm and take a 100µL sample every hour.<br>
-                                            For each sample:
-                                            <ol type="i">
-                                                <li>Make a series of dilutions in 1/10 steps until reaching a 1/10<sup>6</sup>. It can be done in a 96 well plate by adding 10µL of the previous dilution in 90µL of LB medium.</li>
-                                                <li>Plate in agar plates 20µL of dilutions 1/100, 1/10<sup>3</sup>, 1/10<sup>4</sup>, 1/10<sup>5</sup>, 1/10<sup>6</sup>.</li>
-                                            </ol>
-                                        </li>
-                                        <li>Incubate at 37ºC overnight.</li>
-                                        <li>Hopefully, in some of the dilutions the colonies will be countable. Count colonies in plates for at least one dilution of each strain and time point to make a growth/viability curve.</li>
-                                    </ol>
-                                </div>
-                                <div role="tabpanel" class="tab-pane" id="plate">
-                                    <h2>Plate reader</h2>
-                                    <ol>
-                                        <li></li>
-                                    </ol>
-                                </div>
                                  <div role="tabpanel" class="tab-pane" id="Test">
                                      <h2>Silicic acid test</h2>
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-                            </div>
-                        </div>
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-                                <div role="tabpanel" class="tab-pane" id="Test">
-                                    <h2>Silicic acid test</h2>
-                                    <p>For a 1 mL sample:</p>
-                                    <ol>
-                                        <li>Add 20µL of reagent Si-1 and mix.</li>
-                                        <li>Wait for 3 min.</li>
-                                        <li>Add 20µL of reagent Si-2 and 100µL of Si-3 and mix.</li>
-                                        <li>Wait for 10 min.</li>
-                                        <li>Measure absorbance at 824nm.</li>
                                      </ol>
                                  </div>

Difference between revisions of "Team:TU Delft/Notebook"

Revision as of 07:41, 13 October 2016

Notebook

Day Notes

June

29th June 2016

30th June 2016

July

4th July 2016

5th July 2016

6th July 2016

7th July 2016

8th July 2016

11th July 2016

12th July 2016

Nanodrop

13th July 2016

14th July 2016

15th July 2016

18th July 2016

19th July 2016

20th July 2016

21st July 2016

22nd July 2016

23rd July 2016

24th July 2016

25th July 2016

27th July 2016

28th July 2016

29th July 2016

30th July 2016

31st July 2016

August

2nd August 2016

Nanodrop

Nanodrop

3rd August 2016

Lengths of inserts and backbones.

4th August 2016

5th August 2016

Concentrations of plasmid isolations.

9th August 2016

Nanodrop

Nanodrop

10th August 2016

Nanodrop

Nanodrop

Blunt-end ligation protocol

11th August 2016

Nanodrop

12th August 2016

Nanodrop

13th August 2016

14th August 2016

Nanodrop

13th August 2016

15th August 2016

Nanodrop

16th August 2016

17th August 2016

18th August 2016

19th August 2016

22nd August 2016

Nanodrop

23rd August 2016

Nanodrop

24th August 2016

Nanodrop

25th August 2016

Nanodrop

26th August 2016

28th August 2016

Nanodrop

29th August 2016

Nanodrop

30th August 2016

31st August 2016

Nanodrop

September

1st September 2016

29^th June 2016

30^th June 2016

4^th July 2016

5^th July 2016

6^th July 2016

7^th July 2016

8^th July 2016

11^th July 2016

12^th July 2016

13^th July 2016

14^th July 2016

15^th July 2016

18^th July 2016

19^th July 2016

20^th July 2016

21^st July 2016

22^nd July 2016

23^rd July 2016

24^th July 2016

25^th July 2016

27^th July 2016

28^th July 2016

29^th July 2016

30^th July 2016

31^st July 2016

2^nd August 2016

3^rd August 2016

4^th August 2016

5^th August 2016

9^th August 2016

10^th August 2016

11^th August 2016

12^th August 2016

13^th August 2016

14^th August 2016

13^th August 2016

15^th August 2016

16^th August 2016

17^th August 2016

18^th August 2016

19^th August 2016

22^nd August 2016

23^rd August 2016

24^th August 2016

25^th August 2016

26^th August 2016

28^th August 2016

29^th August 2016

30^th August 2016

31^st August 2016

1^st September 2016

2^nd September 2016

3^rd September 2016

6^th September 2016

8^th September 2016

9^th September 2016

10^th September 2016

11^th September 2016

12^th September 2016

13^th September 2016

14^th September 2016

15^th September 2016

16^th September 2016

18^th September 2016

19^th September 2016

20^th September 2016

21^st September 2016

22^th September 2016

23^th September 2016

24^th September 2016

26^th September 2016

27^th September 2016

28^th September 2016

29^th September 2016

30^th September 2016

4^th October 2016

Transformation (OneShot^® Top10 Chemically competent cells)

Colony PCR (Q5^® High-Fidelity 2X Master Mix)