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.

María

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

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.

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.

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.

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.

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.

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.

Lycka

Measure DNA concentration of biobricks from registry by nanodrop.

Nanodrop

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.

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.

Lara

LB agar 14.24 g/400ml; made 2 batches autoclaved.

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.

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.

Lara

Poured plates 400 µL Amp + 400 ml LB agar
15 LBamp & 14LBCam 400 µL Cm + 400 ml LB agar

Iris

Purification IDTs
IDT 1-12, 50 µL Mem
20 µL Nuclease free water

Lycka

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

Celina

Transformation interlab studies.

1. Test device 1
2. Test device 2
3. Test device 3
4. Positive control
5. Negative control

All with 1 µ L BL 21 and 950 µL SOC
Spin down and remove 500 µL and resuspent 50 µL cn plates

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.

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.

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.

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.

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.

Lycka

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

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.

Celina

Transformations

1. mKate – psB1c3
2. mVenus – psB1c3
3. mCerulean – psB1c3
4. Lacl – psB1c3
5. Phap – psB1c3
6. JNP_Sil_dcm psByA5
7. psB1C3
8. psB4A5
9. MQ (100 – µl) plate twice

Resuspend
Spinning down 4000 – 3min
Didn’t add competent cells, so again next page.

Afternoon transformations.

Transformations

1. mKate – psB1c3
2. mVenus – psB1c3
3. mCerulean – psB1c3
4. Lacl – psB1c3
5. Phap – psB1c3
6. JNP_Sil_dcm psByA5
7. psB1C3
8. psB4A5
9. MQ (100 – µl) plate twice

1-8 : – 24µl one shot top 10, 10 µl DNA 250 µl LB, total 277 µl

Spin down 4000 8 min
Take away 200µl
Resuspend
Plate 90 µl
Names plate: correct
Number plates: morning numbers

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.

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.

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.

Iris

Miniprep
1.5µl psB1C3 RFP \( \times \) 2 , centrifuge 3 min max speed
3 tubes (75 – 77 ) with this plasmid

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.

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.

Tessa

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

Nanodropped PCR products.

Nanodrop

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

Tessa

PCR
Amplify E0840 out of backbone
E0840 (GFP) concentration: 126 ng/µl - 2µl
Dissolving primer E080_FW in 247 µl MQ
Working stock 3 is 10 µl of this + 90µl MQ

Component – 50 µl reaction
Nuclease free water – 31.5 µl
5 \( \times \) Phusion HF Butter NEB – 10 µl
10 µM dNTPs promega - 1µl
10 µMVR primer – 2.5 µl
10 µM E0840_FW primer TM 63.1 C – 2.5µl
Template DNA – 2 µl
Phusion DNA Polymerase NEB 0.5 µl
Total 50 µl

PCR
Step at temp(Celsius) for time
Initial denat at 98 30 sec
25-35 cycles at 98,63,72 10,30,30 sec
Final extentsion at 72 10 min
Hold at 4

Electrophoresis of PCR product 1kB benchtop laffer + 4 \( \times \) GFP PCR product

Use product 2 and 4 for PCR 2.

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.

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.

Tessa

Punfication of PCR products of 02/08
Volume tube:

1. 1 = 41 µl
2. 2 = 43 µl
3. 3 = 44 µl

Accidentally added wash solution in first step, added more bind solution to compensate

Consentration (nanodrop)

1. 1 = 86.6 ng/µl
2. 2 = 86.7 ng/µl
3. 4 = 112.2 ng/µl

Charlotte

Restriction and Ligation.

Added :
30 µl Synthesized mVenus
+ 5 µl NEB Buffer 2.1
+ 1 µl EcoRI – HF
+ 1 µl PStI
+13 µl H2) (MQ)
= 50 µl total

Set to incubate at 10.35 h for 2 hrs at 37 ˚ C
Took out at 12.35 h
Purified with SV Clean – Upsystem kit Acc 10 rpotocol - Final volume 15 50 µl
Nanodrop: Blanked with nuclease free water

• 7.7 µl
• 8.5 µl
• 10 µl
• 7.7µl

So: 7.7 ng/µl

Mode protocol for ligation: water 1.29 µl, total volume 20 µl
Pipetted the scheme, left for incubation at room temperature at 14:20. Started transformation at 17:45.(Lab book Lara)

Tessa

PCB: add promoters to GFP (E08040)

Primers: (template NA: E0840, tube 2)
Reverse: VR

• FW1 : J23100 E0840_FW
• FW2 : J23105 E0840_FW
• FW3 : J23108 E0840_FW
• FW4 : J23113 E0840_FW
• FW5 : J23117 E0840_FW

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.

Tessa

Nanodrop:

1. blank = nuclease free water conc. Ng/ \( mu \) l
2. J23100 E0840 – 69.4
3. J23105 E0840 – 95.9
4. J23108 E0840 – 88.3
5. J23113 E0840 – 97.7
6. J23117 E0840 – 90.3

Restriction (incubated for 2h at 39 ˚ C):

Purification: 50 µl product -> 50 µl membrane bind solution

Nanodrop

1. 0 – blank = nuclease fee water ng/ µl
2. 3.3
3. 3.5
4. 3.5
5. 2.8
6. 3.4

Ligation

Incubated for 3h at about 17 ˚C
Transormation into topio homemade cc cells

1. J23100 E0840 pSB1C3
2. J23105 E0840 pSB1C3
3. J23108 E0840 pSB1C3
4. J23113 E0840 pSB1C3
5. J23117 E0840 pSB1C3
6. positive control – RFP
7. negative control - no DNA, water

Overnight incubating of plates

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.

These DNA samples were sent to sequencing.

Tessa

Another overnight incubation for colony growth 2 developed some RFP colonies

Colony PCR
1-8 J23105 E0840 pSB1C3
9-19 J23108 E0840 pSB1C3

• TempºC - time
• 95 – 15 min
• 95 – 30 sec
• 53 – 30 sec
• 72 – 15 min
• 72 – 5 min
• 4 – inf

DNA Electrophoresis – good colonies
1,2,4,5,8,9,12,(15,16), 17,19

Tessa

Miniprep

1. 1 – 1 – J23105 – E0840 – pSBIC3
2. 2 – J23105 – E0840 – pSBIC3
3. 4 – J23105 – E0840 – pSBIC3
4. 5 – J23105 – E0840 – pSBIC3
5. 8 – J23105 – E0840 – pSBIC3
6. 9 – J23108 – E0840 – pSBIC3
7. 12 – J23108 – E0840 – pSBIC3
8. 17 – J23108 – E0840 – pSBIC3
9. 19 – J23108 – E0840 – pSBIC3
10. mCerulean – J23108 – E0840 – pSBIC3

Added 2 µl culture to 1.5 micro – centrifuge tube (twice 1 µl + centrifuge)
Final solution: 30 µl

Nanodrop ng/µl

1. 156.8
2. 190
3. 102.3
4. 341.3
5. 114.8
6. 116.7
7. 119.8
8. 126.4
9. 198.7
10. 91.3

Sequencing:

1. 3 µl DNA 4.31 µl MQ
2. 2.63 µl 4.87 µl MQ
3. 4.89, 2.61
4. 1.46, 6.04
5. 4.36, 3.14
6. 4.28, 3.22
7. 4.17, 3.36
8. 3.96, 3.54
9. 2.52, 4.98
10. 5.48, 2.02

mCerulean has one silent mutation CCC->UG

Tessa

Cryostocks: 8mL, 8/3 = 2.67 mL fresh medium/glycerol

Liza

Transformation BL21 mCerulean
Plus: RFP inps BLC3 in BL21
Minus: MQ with BL21, m Cerulean in PSBLC3 with BL21 as protocol but step 9 – 90 mins plated on CM plates.

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

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.

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

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.

Liza

Transformation mCerulean

Positive control worked,
Negative control no changes
mCerulean we do have colonies
Colony OCR from transformation 08/08
3: mCerulean pSBLC3

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

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

Nanodrop

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

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

Iris

PhaP – PCR 1, \( 2 \times 50 \mu l \)

• \( 10\mu l \) HF phusion
• \( 10 \mu M \) dNTPs 1 \( \mu l \)
• 10 \( \mu l \) FW (phap - FW) 2.5 \( \mu l \)
• 10 \( \mu l \) RV primer (VR) 2.5 \( \mu l \)
• Phusion polym 0.5 \( \mu l \)
• DNA 1.2 \( \mu l \) 207 ng/ \( \mu l \)
• Nuclease free water: 32.35 \( \mu l\)

Celina and Liza

3-8 mCerulean – 1.5 \( \mu l \) culture in LB, 600 \( \mu l \) MQ
Nanodrop

• 3) 73.7 ng/µL
• 4) 134.4 ng/µL
• 5) 122.4 ng/µL
• 6) 91.3 ng/µL
• 7) 67.4 ng/µL
• 8) 66.0 ng/µL

Cryostocks in 2 \( \mu l \) cryotubes
mCerulean poBLC3, BL21, Colonies: 3 to 8
Sequencing, DNA + MQ in \( \mu l \) with \( VF_2 \) and VR:

• 3) 6.8 + 6.7
• 4) 3.6 + 3.8
• 5) 4.1 + 3.4
• 6) 5.5 + 2
• 7) 7.4 + 0.1
• 8) 7.5 + 0

All sent for sequencing.
All Colonies had mutation: Silent mutation Pro to Pro and CCC to CCG.
Colonies 3 and 4, position 22156.

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

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.

Liza

Colony PCR mVenus PSBLC3 mKate PSB1C3.

Mastermix in \( \mu l \)

• 225 QS HS \( 2\times \) mastermix
• 22.5 primer VF2
• 22.5 primer VR
• 180 Mili Q
• Total 450 \( \mu l \).

Electrophoresis
1 is benchtop 1 LB ladder
5 to 20 are colonies, 1 mKate and 1 mVenus

Tessa

Miniprepped colony 14 and 21 of yesterdays overnight culture.

Nanodropped miniprep product.

Nanodrop

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.

Liza

transformation mVenus, mKate BL21

• mVenus is PSB1C3 BL21
• mKate PSB1C3 BL21
• plus RFP PSB1C3 BL21
• minus sterile MQ BL21

Plated on CM LB plates incubated at 37 degrees overnight, put in the fridge on Saturday

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.

Lara

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

Nanodrop

These plasmids were sent to sequencing.

Charlotte

Nanodrop

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

Liza

Colony PCR

mVenus and mKate 2 more h in stoot . mVenus has 2 color colonies (crème and yellow) then colony PCR of mVenus, mKate. From mVenus take few colonie of both colored.

8 colonies mVenus yellow, 2 of mVenus crème and 8 mKate

PCR mix met Gotaq
100 \( \mu l \) 5 \( \times \) green Gotaq Buffer <\br> \( 19 \mu l \) PCR Nucleotide mix
95 \( \mu l \) primer VF2
4.75 \( \mu l \) Gotaq DNA pdymease
546.25 \( \mu l \) Nuclease free water Total: 950 \( \mu l \), 50 \( \mu l \) per reaction

mVenus: colony 1-8 yellow colonies
Colony 9-10 crème colonies
mKate: colony 1-8 <\br>

Charlotte

Sequencing had the following results:

Liza

Electrophoresis of colony PCR 15/08

1 - benchtop 1 bK ladder
2 t0 11 mVenus colony 1 to 10 <\br> 13-20 mKate colony 1 to 8

Overnight culture – PSBLC3 mVenus colony 2,3 and 8
mKate colony 1,3 and 4
RFP colony 1 (Possitive control 12/08)

Charlotte & Liza

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

Tessa

Miniprep
\( 2 \times 1 ml \) culture pre colony

1. mKate c1
   
2. mKate c3
   
3. mKate c4
   
4. mVenus c2
   
5. mVenus c3
   
6. mVenus c8
   
7. RFP c1

Nanodrop ng/µL

1. 264.0
   
2. 129.2
   
3. 271.4
   
4. 215.7
   
5. 376.1
   
6. 199.3
   
7. 163.4
   

Tessa

Transformation of the InterLab Study plasmids into BL21.

Liza

Recieved purified GFP µM
Aliquot 5 15 µl -80 degrees fridge.

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.

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

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.

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

These plasmids were sent to sequencing.

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

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

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.

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

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.

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.

Lara

Plasmid isolation was performed for the K1149051 pSB4A5 BioBrick.

Nanodrop

These plasmids were sent to sequencing and were sequence confirmed.

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

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.

Lara

Prepared LB Amp+Cm plates.

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.

Lara

Transformations into BL21 were performed for K1149051 pSB4A5, J23100 phaP pSB1C3 + K1149051 pSB4A5, J23105 phaP pSB1C3 + K1149051 pSB4A5, J23108 phaP pSB1C3 + K1149051 pSB4A5, J23113 phaP pSB1C3 + K1149051 pSB4A5 and J23117 phaP pSB1C3 + K1149051 pSB4A5. RFP pSB4A5 and RFP pSB4A5 + RFP pSB1C3 were used as positive controls and MilliQ as a negative control. In total 70 µL was plated on selective LB plates and they were incubated at 37 degrees overnight.

Cryostocks were made for K1149051 pSB4A5 in Top10 cells.

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

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

Lara

Yesterday's transformations in BL21 did not yield colonies for K1149051 pSB4A5 and for J23100 phaP pSB1C3 + K1149051 pSB4A5. These were transformed again, together with BolA_con pSB4A5, K1149051 pSB4A5 and RFP in pSB4A5, pSB4A5 and pSB1C3 as positive control and MilliQ as negative control. These transformations were plated on selective LB plates and stored overnight at 37 degrees.

For the Gibson Assembly, new plasmid needed to be isolated.

Nanodrop

A colony PCR with GoTaq buffer was performed for the different promoters of phaP pSB1C3 + K1149051 pSB4A5.

Colonies 2, 3, 4, 8, 9, 10, 13, 14, 15, 18, 19 and 20 were transferred into liquid LB+Amp+Cm and stored overnight at 37 degrees.

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.

Charlotte

Cryostocks: Were made for colonies 4, 10, 13 and 18 for the different promoters of phaP pSB1C3 + K1149051 pSB4A5. They were stored at -80 degrees.

Liquid cultures: Each colony of BL21 transformation should be good. Liquid cultures were made of BolA_con pSB4A5, K1149051 pSB4A5 and J23100 phaP pSB1C3 + K1149051 pSB4A5 and stored at 37 degrees to be used tomorrow to make cryostocks.

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

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.

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.

Lara

Doing things for Lycka and María: Plate was removed from plate reader and file was saved. Platereader plate was stored at 4 degrees.

mVenus pSB1C3 was miniprepped and stored at -20 degrees.

Nanodrop

A Phusion PCR was performed again for Gibson Assembly. The vector for mKate and mVenus and the insert for GFP yielded a correct PCR product and were gel isolated.

Nanodrop

Since there are an insert and a vector present that are compatible for mKate and K1149051 pSB4A5, these were Gibson Assembled according to the protocol and transformed into Top10 cells. As a negative control for the Gibson Assembly, NF water was used instead of the vector. Transformations were plated on selective LB plates by Lycka and stored overnight at 37 degrees.

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.

Lycka

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

Lara

For the Gibson Assemblies, we still needed the mCerulean vector, the GFP vector and the mVenus insert. For this, another phusion PCR was performed and it ran overnight.

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.

Lara

The phusion PCRs of yesterday were gel-isolated. Since mVenus once again did not yield a proper product, we decided to discard this part.

Nanodrop

A Gibson Assembly reaction was performed according to the protocol for the GFP + K1149051 assembled plasmid and the mCerulean + K1149051 assembled plasmid. After 60 minutes of incubation at 37 degrees, they were transformed into Top10 cells. For this, RFP pSB4A5 was used as a positive control and vector without inserts were used as negative controls. Transformation products were plated on selective LB plates and stored overnight at 37 degrees.

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.

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.

The transformations from the Gibson Assembly still did not yield any colonies.

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.

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.

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.

Tessa

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

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.

Lycka

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

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.

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.

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.

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.

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.

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

Lycka

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

OD600 measurements

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

María

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

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.

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

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

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

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

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

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

María

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.

Lara

The PCR product of BolA of María was gel purified and blunt end ligated. Afterwards a transformation was performed into Top10 cells with RFP pSB1C3 as positive control and MilliQ as negative control. Transformations were plated on LB+Cm plates and stored overnight at 37 degrees.

Lara

Plates of yesterday's transformations were transferred from the 37 degrees incubator into the fridge.

Célina

Colony PCR

Length: 754 for 317 min
Primes: Addgene_col_JW and Addgene_Cd_RV

Samples: 25
HJ \( 2\times \) master mix \( 325 \mu l \)
JW primer \( 32.5\mu l \)
RV primer \( 32.5\mu l \)
miliQ \( 260\mu l \) (Nuclease free water)
TOTAL: \( 650 \mu l \)

Dreamtaq
For 1: 12.5 \( \mu l \) + 1 \( \mu l \) + 1 \( \mu l \) + 5.5 \( \mu l \) = 20 \( \mu l \)

Annealing temperature I \( 57 \degree C \)
Rep: 29
Last: \( 72 \degree C \) for 10 min

\( 20 \times 2 \mu l \) Loading buffers

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.

Cryostock

Colonies: 1, 2, 5, 6, 16, 17.
Cell type: Top 10
Biobrick: BolA
Backbone: pBbASC
In plastic bag

Celina

Miniprep

Steps 1-13 done
1,4: NF Water and they are at \( -20 \degree C \) fridge labeled with no number yet, in a yellow box.

Lycka

Transformation to BL21 of BolA ind (corrected sequence, fixed by PCR). Tube 3 from minipreps Célina (yesterday) was used to transform.

• Bola ind (CM)
• BolA ind + OmpA Sil Taur (CM + AMP)
• MiliQ - control
• RFP pSB1C3 + control

Lara

Restriction of plasmids to put the inserts into the standard backbone from the Addgene backbones.

Nanodrop

Since the amount left of BolA and RFP pSB1C3 was too little, these needed to grow from cryostocks again to be miniprepped.

Restrictions were performed and the backbone was gel-isolated; the rest did not yield restriction products visible on the electrophoresis gel.

Lycka

Inoculate cultures in fresh LB for growth study and confocal. ODs measured at 600nm in spectrophotometer.

OD600

Cryostocked OmpA Sil Taur + BolA ind and BolA ind in BL21.

Lara & Lycka

Tried miniprepping straight from the cryostocks to yield more isolated plasmids.

Nanodrop was performed by Lycka.

Nanodrop

Lycka

Pour plates with LB + CM and AMP.

Inoculate for SEM, TEM, confocal and solar cell experiments.

• OmpA Sil Taur
• INP Sil Sdom
• Sil Sdom
• BolA ind
• BolA ind + OmpA Sil Taur

Lycka

Restriction of Sil_Sdom, OmpA_Sil_Taur, INP_Sil_Sdom, BolA_con, BolA_ind with EcoRI and PstI to put in standard backbone. Load restriction product on gel. Run at 100 mA for 45 min. No bands visible.

Try to PCR with standard primers and digest the PCR product. Phusion Polymerase used. Annealing temp 56 degrees; extention time 90 sec. 5µL PCR product was put on gel.

Purification and nanodrop done by Charlotte. Respective concentrations:

• 124.3 ng/µl
• 89.2 ng/µl
• 96.4 ng/µl
• 104.6 ng/µl
• 135.8 ng/µl

Lara

To test which settings for UV sterilization are effective, different powersettings within the Stratagene UV Stratalinker 1800 were used. 700 µL of cells transformed with BolA_con in pSB4A5 were loaded in an eppendorf tube and were separately treated with the lid open, so the UV light could be transmitted. The used powersettings were 100000µJ, 120000µJ, 140000µJ, 160000µJ, 180000 µJ, 200000 µJ, 220000µJ and 240000 µJ. Afterwards 50 µL was plated on LB+Amp plates.

Lycka

Restriction of PCR products of Sil_Sdom, OmpA_Sil_Taur, INP_Sil_Sdom, BolA_con, BolA_ind with EcoRI and PstI. Gel purify BolA ind, because its current backbone has the same resistance as the standard backbone (chloramphenicol). Normally purify others (they come from an amp resistant backbone). Concentrations after purification:

• 40.8 ng/µl
• 22.6 ng/µl
• 34.8 ng/µl
• 36.0 ng/µl
• 5.0 ng/µl

Ligation of 100 ng vector with respective inserts. 1:3 molar ratio used. Incubated for 3 hours.

Transformation to TOP10. Plated after 1.5 hour incubation in nonselective LB. Plated on LB CM plates.

Lara

Checked if there were colonies after UV sterilization. Colonies did appear. We figured that the UV light was not transmitted through the entire layer of 700 µL of cells, so I actually plated non sterilized cells. For that, a new method was used.

New method UV sterilization: Plating 30 µL BolA pBbA5C transformed cells directly on selective LB plates and sterilizing these with the recommended powersetting of 120000µJ and 240000µJ. As positive controls 500 µL cells were washed 3 times with ethanol or heated at 95 ºC for 10 minutes; as negative control living cells were plated. All were incubated at 37 ºC overnight.

Solar cell experiment preparation: First, all cell pellets were diluted as such that same cell concentrations were yielded. Afterwards, these cells were UV sterilized 3 times with a power of 240000 µJ.

Lara

Checked if there were colonies after UV sterilization. No colonies appeared in both powersettings.

Lara & Giannis

Solar cell experiment: Visited Stefaan Heirmans at the TU Delft to properly test the change in efficiency in solar cells, using a solar simulator. A total of 3 µL was loaded onto the solar cell and the efficiency was measured.

Solar cell efficiency

Since the bubble of liquid also worked as a lens, we figured these were not the most conclusive results. For that, we have decided to redo these experiments, with cells dried onto glass plates.

Lara

Plasmid isolation of Sil_Sdom, OmpA_Sil_Taur, INP_Sil_Sdom, BolA_con, BolA_ind in the standard backbone.

Nanodrop

These plasmids were sent to sequencing.