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Team:Warsaw/Wetlab
Week 1 (25.06-29.06)
Monday:
We entered the lab and got the general idea of where things were and how we are going to work there. We had a talk with our instructors on how to divide the work, what we have to prepare first and what we will need in the future. We started off with locating two constructs with listeriolysin.
Tuesday:
As an introduction to the wetlab work, we prepared the competent E. coli strains: TOP10 – 80 eppendorfs, BL1 – 40 eppendorfs, and MC1061 – 40 eppendorfs.
We tried a control transformation of all three strains: TOP10, BL21, and MC1061 with 2 ul of DNA.
We also prepared plates with ampicillin and retrieved GFP, RFP and YFP from the distribution plates.
Wednesday:
Again, we transformed TOP10, BL21, and MC1061 strains of competent E. coli, this time with 5 ul of DNA each.
We transformed TOP10 strain with the construct with lysteriolysin, as well as prepared overnight cultures for MIC calculation. We also prepared plates with LB agar.
Thursday:
MIC calculations.
Friday:
Bacteria transformed with listeriolysin construct formed colonies – overnight cultures prepared.
Week 2 (2.07-6.07)
Monday:
We tried our first PCR without template – since we didn’t know which concentration of primers will work best, we tried 3 ul and 5 ul.
Total volume: 20 ul
Taq mastermix: 10 ul
Primers: 5 ul each and 3 ul each (+4 ul nuclease free water)
Tuesday:
Electrophoresis of our PCR products: we had products in both! We proved our new technique works! We had significantly more product where we used higher concentration of primers, so we used 5 ul of primers in our following PCRs.
Wednesday:
One of our instructor conducted Clone Manager workshops. We learned how to use the software and how to create and modify DNA molecules, how to design primers for the existing ones and how to check restriction sites – e.g. which enzyme to use when.
Thursday & Friday:
Clone Manager work! Preparing primers to produce our new parts (through PCR without template of course) – promoter, RBSes and terminator for B. subtilis.
Week 3 (9.07-13.07)
A little more Clone Manager work, as well as ordering synthesis of our primers and waiting for their arrival in our lab!
Week 4 (16.07-20.07)
Tuesday:
Our new primers finally arrived on our lab tables! We start off with ‘no template PCR’ of our new promoter and RBS.
Wednesday:
We ran electrophoresis of yesterday’s PCR products, and guess what? Again we were successful!
So we ran more ‘no template PCRs’ – this time more RBSes and terminator.
Thursday:
We ran clean up of our PCR products, and checked their concentrations on NanoDrop spectrophotometer – the results were not that good, the concentrations were not high.
Friday:
Despite the low concentration of our parts, we decided to digest the promoter and GFP with EcoRI+SpeI, RBSes and terminator with XbaI+PstI
Then we started ligation: promoter with RBSes and GFP with terminator. Thanks to our FastDigest enzymes, both the digestion and ligation lasted much shorter than normally – one hour. We also ran the ligation in 4 °C for longer periods like over night or even over weekend.
Week 5 (23.07-27.07)
Monday:
We ran electrophoresis of the products of ligation, to check whether everything went fine.
Tuesday:
We ran another set of PCRs to get some more of our parts.
Wednesday:
We repeated ligation of our parts – the promoter with RBS.
Thursday:
We decided to run standard PCR of our ligation products: e.g promoter+RBS (left primer of promoter and right of RBS)
Friday:
another day of running PCRs (this time with template).
Week 6 (30.07-3.08)
Monday and Tuesday:
We checked the concentration of DNA in our PCR products by spectrophotometry (NanoDrop), some of the concentrations were very satisfactory, some were still pretty low.
Wednesday:
Digesting and electrophoresis to check how the ligation went on. Then more Clone Manager work!
Week 7 (6.08-10.08)
Monday:
Electrophoresis and pJET cloning, to multiply our parts on a plasmid.
Tuesday:
We decided we need to make 100% sure if the sequence of our parts didn’t mutate somewhere along the way (during all the PCRs, digestions, ligations, cloning, transformation, and all), so we prepared samples of our parts with pJET primers and sent them to Oligo – company sequencing DNA, which is also one of our sponsors.
Wednesday:
Digestion of RBS, GFP and plasmids pSB1K3 and pSB1C3. Then of course ligation of our parts: RBS+GFP+pSB1C3 and RBS+pSB1K3. Then TOP10 transformation.
Thursday:
Overnight cultures of our transformants. No template PCR of our promoter.
Friday:
Isolation of plasmids ligated on Wednesday (RBS+pSB1K3 and RBS+GFP+pSB1C3)
Week 8 (13.08-17.08)
Monday:
We sent some more samples to sequencing. We also checked the concentration of the PCR product from Thursday (on NanoDrop).
Also, overnight digestions of RBS and pSB1K3 were set.
Tuesday:
We ran PCR of pCEP – a mammalian plasmid which contained oriP, but not in BioBrick format. We designed primers which would help us to eliminate illegal restriction site in the sequence. We also ran PCR to check whether our RBS on pSB1K3 has a correct sequence.
Some more digestions: RBS – EcoRi+PstI, RBS – EcoRI+SpeI, pSB1C3&pSB1K3 – EcoRI+PstI, GFP – XbaI+PstI. Then we ligated RBS with pSB1C3, RBS with pSB1K3, and RBS+GFP+pSB1C3. Then we ran transformation of TOP10 strain of E.coli with our ligation products.
Wednesday:
We prepared overnight cultures of the ligation products we transformed on Tuesday. Then, we ran PCR to confirm correctness of our ligation samples with Phusion polymerase. Unfortunately, after running electrophoresis, we had a very unpleasant smear on the gel, and we decided to repeat the PCR with Taq polymerase.
We also prepared another set of competent E. coli – TOP10 strain. This time we had 90 eppendorfs.
Thursday:
Isolation of Tuesday’s ligation products. Our pSB1K3 stock ended. So we decided to cut it with XbaI and SpeI of a sample of RBS+pSB1K3, then run electrophoresis and gel out empty plasmid, and then run autoligation and transformation.
We also ran another set of ‘no template PCRs’, this time with Phusion polymerase, as well as checked concentration of our today’s isolated plasmids on NanoDrop – they were pretty much satisfactory.
Friday:
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Digestion and ligation of RBSes+pSB1K3, P+pSB1C3, P+RBS+GFP+pSB1K3. Transformation on TOP10.
We ran electrophoresis with yesterday’s isolated plasmids to check to presence of our input.
Week 9 (20.08-24.08)
Monday:
We cloned our parts on pJET plasmid using pJET cloning kit and prepared overnight cultures of Friday’s transformation.
Overnight Bacillus subtilis cultures.
Tuesday:
Bacillus cultures didn’t grow up. We decided to leave them for longer. Isolation of the pJET plasmids that were transformed yesterday. Construction of plasmid: RBS+GFP+pSB1C3 (digestion, ligation, transformation).
Checking concentration of isolated plasmids on NanoDrop.
PCR on pCEP to get oriP.
Deep freeze of Bacillus cultures.
Wednesday:
Isolation of plasmids from the previous day and preparing them for sequencing. Overnight cultures of RBS+GFP+pSB1C3. Digestion of pTG262 (EcoRI+PstI) and P+RBS+GFP, ligation and transformation. NanoDrop checking of concentration of isolated plasmids.
Thursday:
Transformation of B. subtilis.
Isolation of plasmid DNA from the previous day’s overnight cultures, then checking the concentration on NanoDrop.
Digestion of promoter, RBS+GFP, pSB1K3 – then overnight digestion of constructs P+RBS+GFP+pSB1K3.
Creation of LLO construct: digestion: RBS, LLO, pSB1C3. Inoculation of transformants from the previous day. Ligation and transformation of constructs: RBS+GFP+pSB1C3.
Week 10 (27.08-31.08)
Monday:
Overnight cultures of the transformants which resembled growth. None of the transformants with the LLO construct formed colonies – no growth – something is wrong with our construct!
Repetition of the whole procedure of preparing the LLO construct and transformation.
Reviving of BioBricks (mammalian RBSes from the distribution) J63003 and K165002, transformation and inoculation. PCR for pCEP – to get oriP.
Tuesday:
Isolation of the plasmid DNA from last day’s overnight cultures. Preparing overnight cultures of the mammalian RBSes.
Transformants with LLO didn’t grow up. Control digestion of the LLO part and electrophoresis to check what’s wrong. Also electrophoresis of the PCR products.
Wednesday:
Isolation of the plasmids with mammalian RBSes.
Digestion of the mammalian RBSes, pSB1C3 and RBS+GFP+pSB1C3.
Ligation of mammalian RBSes+RFP+pSB1C3, then transformation and inoculation. We also ran out of stock of pSB1C3, and so we digested it with XbaI+SpeI to run autoligation and transformation.
Thursday:
Overnight batch cultures of transformants from the previous day.
Friday:
Isolation of plasmid DNA from yesterday’s batch cultures and NanoDrop checking the DNA concentration.
Week 11 (3.09-7.09)
Monday:
Inoculation of SuperFolder GFP.
Tuesday:
Isolation of plasmid DNA from last day’s cultures. DNA concertation check.
Electrotransformation of pTG262 into E. coli. Preparation of overnight culture of Bacillus subtilis for the next day transformation. We also ran PCR on pCep with a new pair of primers.
Wednesday:
Control digestion of constructs and electrophoresis to assure our parts are correct.
Transformation of B. subtilis with pUB110 (control) and pTG262.
Thursday:
Isolation of pTG262 from E. coli. Then we transformed B. subtilis with pTG262 islotated from E. coli.
Digestion of all previous constructs and preparation of a full construct – ligation P+RBS+GFP+pSB1C3, subsequently transformation and inoculation.
Friday:
Electrophoresis of digestions from the previous day – wrong placements of inserts. Inoculation of transformants for overnight culture. Transformation of pTG262 into E. coli TOP10, incubation during the weekend.
Week 12 (10.09-14.09)
Monday:
Isolation of plasmid DNA from 7th Sept. NanoDrop measuring of DNA concentration. Then preparation of samples for sequencing. Then we digested P+RBS+GFP+pSB1C3 construct and ran electrophoresis to check the placement of the insert.
Tuesday:
Gel-out digestions from the previous day.
Wednesday:
PCR of pCep4 – changes in gradient annealing temperatures.
Digestion of all our parts, ligation into pSB1A3 and pSB1C3, overnight ligation.
Thursday:
Preparation of samples for DNA sequencing.
Digestion of our parts, ligation and transformation.
Friday:
Isolation and clean up of the plasmid DNA from the previous day.
Week 13 (17.09-21.09)
Putting the final constructs together! Last preparations before shipping our parts to Boston!
Week 14 (24.09-28.09)
Wiki!
