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Our lab
We have our own iGEM TU Delft lab in the new Applied Science building on the edge of the TU Delft campus. It is classified as an ML-1 lab, the lowest safety level to work with modified organisms, which is enough for our experiments. Apart from this lab, we are also working in an optical lab, which is also ML-1 classified. In here built our own laser set-up.
![](https://static.igem.org/mediawiki/2016/4/40/T--TU_Delft--Our_Lab.png)
Our office
Our office is our homebase for when we're not working in the lab. Here we work on things like the safety tool, the wiki, the modeling, and processing our results. This summer the Bionanoscience department moved to a new building, so it took some time before we had our own office. When we finally got our office, we quickly made it our home. Next to our office, there is a meeting room, where we have a weekly meeting with our TA’s and PI’s to keep everyone up to date and discuss problems we might encounter.
![](https://static.igem.org/mediawiki/2016/7/79/T--TU_Delft--Our_Office.jpg)
Lab safety
Our lab is classified the lowest safety level (level 1), meaning that our experiments involve low to no risk. All the members of the team have successfully completed the following safety tests: Lab safety test General safety test of the building we currently work in Biological safety test for ML-1 lab General safety test of the building we worked in before June All the members of the team have received safety training, including: Introduction to sterile working General lab training (using a PCR machine, making gels, etc.) General safety information, regarding contact persons and locations The safety of our experiments was supervised by Erwin van Rijn (Safety Manager of the lab) and Jeremie Capoulade (Safety Manager of the lasers). The supplies we needed in the lab were provided with the help of our instructor Esengül Yildirim. The research has been conducted with respect to the regulations of biosafety for The Netherlands, that can be found here.
Day Notes
29th 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.
30th 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.
4th 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.
5th 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.
6th 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.
7th July 2016
María
Transformation of same BioBricks as on 5th July using a different transformation protocol since we believed that the negative result was due to a mistake during transformation.
8th 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.
11th 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.
12th 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.
13th 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.
14th 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.
15th 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.
18th July 2016
Lycka
Restriction of backbones pSB1C3 and pSB4A5 with EcorI and PstI. Gel electrophoresis of digested fragments. Gel picture yielded no bands.
19th 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 14th of July: INP_Sil_Sdom in pSB1C3 and BolA_ind and BolA_con in both pSB1C3 and pSB4A5.
20th July 2016
Lycka and Tessa
Transformation of plasmids ligated by Maria (19th of July) together with the ones ligated on 14th 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.
21st July 2016
María
Colony PCR of yesterday's transformation results from Lycka and Tessa. However, not all transformations yielded colonies.
![electrophoresis](https://static.igem.org/mediawiki/2016/1/1a/T--TU_Delft--Electrophoresis_20160721_1.png)
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.
22nd July 2016
María
Continue with colony PCR of 20th of July transformation results from Lycka and Tessa.
![electrophoresis](https://static.igem.org/mediawiki/2016/0/0b/T--TU_Delft--Electrophoresis_20160722.png)
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.
23rd July 2016
María
Continue with colony PCR of yesterday's transformation results from Lycka.
![electrophoresis](https://static.igem.org/mediawiki/2016/8/8d/T--TU_Delft--Electrophoresis_20160723.png)
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 21st, 22nd and 23rd of July and transfer to selective LB according to the resistance expressed by each plasmid.
24th 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.
25th 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.
27th July 2016
Lycka
Colony PCR of plates transformed yesterday.
![electrophoresis](https://static.igem.org/mediawiki/2016/b/bf/T--TU_Delft--Electrophoresis_20160727_1.png)
![electrophoresis](https://static.igem.org/mediawiki/2016/e/e2/T--TU_Delft--Electrophoresis_20160727_2.png)
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.
28th 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.
29th July 2016
Lycka and Célina
Repeat of the colony PCR of plates from the 26th of July that yielded a negative result.
![electrophoresis](https://static.igem.org/mediawiki/2016/0/02/T--TU_Delft--Electrophoresis_20160729.png)
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.
30th July 2016
Lycka
Colony PCR of OmpA_Sil_Sdom in both backbones from the 29th of July. The PCR machine was filled with colonies from older plates which had not yielded any good colonies yet: mKate (26th of July), mKate (mKate 30th of June), INP_Sil_Sdom (20th of July).
![electrophoresis](https://static.igem.org/mediawiki/2016/2/2f/T--TU_Delft--Electrophoresis_20160730.png)
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 29th of July. Cryostocks stored at -80 degrees, isolated plasmid stored at -20 degrees.
31st 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.
2nd 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.
![electrophoresis](https://static.igem.org/mediawiki/2016/9/98/T--TU_Delft--Electrophoresis_20160802.png)
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 |
3rd 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.
4th 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 |
5th 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.
10th 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 4th 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.
11th August 2016
Tessa
Colony PCR'd colonies from yesterdays transformation using Q5® mastermix.
Ran a 1% agarose gel of the PCR product.
![electrophoresis](https://static.igem.org/mediawiki/2016/d/d5/T--TU_Delft--Electrophoresis_20160811.png)
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.
12th 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.
13th 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.
14th 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.
18th August 2016
Tessa
Transformation of the InterLab Study plasmids into BL21.
19th 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.
22nd August 2016
Tessa
Transformation of the InterLab Study plasmids into Top10.
Colony PCR of J23100 E0840 pSB1C3 using GoTaq®. Plate from 11th August.
Ran a 1% agarose gel to verify part size
![electrophoresis](https://static.igem.org/mediawiki/2016/a/a1/T--TU_Delft--Electrophoresis_20160822.png)
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.
23rd August 2016
Tessa
Colony PCR of the InterLab Study plates using DreamTaq®. Picked eight colonies of each plate.
Cryostocked J23100 E0840 pSB1C3 in Top10.
Ran a 1% agarose gel of the InterLab Study colony PCR products.
![electrophoresis](https://static.igem.org/mediawiki/2016/4/46/T--TU_Delft--Electrophoresis_20160823.png)
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.
24th August 2016
Tessa
Put InterLab Study cultures in fridge for later use.
25th August 2016
Tessa
Resuspended FITC according to InterLab protocol.
29th August 2016
Tessa
Transformed J23100-, J23105-, J23108-, J23113- and J23117 E0840 pSB1C3, and BolA_con pSB4A5 into BL21.
30th 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®.
Ran a 1% agarose gel of PCR products to verify part size.
![electrophoresis](https://static.igem.org/mediawiki/2016/3/39/T--TU_Delft--Electrophoresis_20160830.png)
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.
31st August 2016
Lycka
Electrophoresis of PCR fragments yesterday.
![electrophoresis](https://static.igem.org/mediawiki/2016/7/79/T--TU_Delft--Electrophoresis_20160831_1.png)
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.
![electrophoresis](https://static.igem.org/mediawiki/2016/9/9a/T--TU_Delft--Electrophoresis_20160831.png)
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.
1st 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
![electrophoresis](https://static.igem.org/mediawiki/2016/9/91/T--TU_Delft--Electrophoresis_20160901_2.png)
Lycka
Electrophoresis of overnight PCR for lineralization.
![electrophoresis](https://static.igem.org/mediawiki/2016/2/2a/T--TU_Delft--Electrophoresis_20160901.png)
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.
2nd 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.
3rd 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.
6th September 2016
Lycka & Lara
Colony PCR for the transformants of the Gibson Assembly.
![electrophoresis](https://static.igem.org/mediawiki/2016/b/b0/T--TU_Delft--Electrophoresis_20160906.png)
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.
8th September 2016
Lycka
Miniprep, cryostock and prepare for sequencing colonies 43 and 44. The concentrations were 252.6 and 242.6 ng/µl, respectively.
9th 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.
11th September 2016
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.
12th September 2016
María
Colont PCR of transformations from yesterday 16 colonies of each transformation were picked, but only analized 15 of each.
![electrophoresis](https://static.igem.org/mediawiki/2016/0/06/T--TU_Delft--Electrophoresis_20160912_1.png)
![electrophoresis](https://static.igem.org/mediawiki/2016/8/81/T--TU_Delft--Electrophoresis_20160912_2.png)
![electrophoresis](https://static.igem.org/mediawiki/2016/1/13/T--TU_Delft--Electrophoresis_20160912_3.png)
Red arrows point at colonies picked for overnight cultures in selective medium.
13th 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 sequencies for OmpA_Sil_Sdom contained a strange long insert, this silicatein gene version was dropped for further experiments.
15th September 2016
María
Transformation of plasmids with confirmed sequences from 13th 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.
20th September 2016
María
Transfer of colonies from 15th of September transformations into overnight culture for cryostocking.
21st 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.
Protocols
During our lab work we used the following protocols.
- Chemically competent Cells
- PCR (Phusion)
- Gel Isolation (From Promega Wizard™ Kit)
- Restriction (NEB enzymes)
- Ligation
- Transformation (TOP10)
- Transformation (BL21)
- Transformation (OneShot® TOP10)
- Colony PCR (Q5® High-Fidelity 2X Master Mix)
- Colony PCR (GoTaq)
- Colony PCR (DreamTaq)
- DNA Electrophoresis
- Overnight Cell Cultures
- Cell Frozen Stock
- Plasmid Isolation (From Promega PureYield™ Plasmid Miniprep Kit)
- Restriction Product Purification (From Promega Wizard™ Kit)
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).
Recipe for TSS (100 mL):
- 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 - 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 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.
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:
- 1µL of plasmid or 2µL of ligation product.
- 20µL 5X KCM buffer.
- 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 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.
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.