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Team:Warsaw/Wetlab
Week 1 (25.06-29.06)
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. We prepared the competent E. coli strains: TOP10 – 80 eppendorf tubes, BL21 – 40 eppendorf tubes, and MC1061 – 40 eppendorf tubes. We tried a control transformation of all three strains with 2 ul and 5 ul of DNA. We also prepared plates with ampicillin and retrieved GFP, RFP and YFP from the distribution plates. We also transformed TOP10 strain with the construct with listeriolysin (BioBrick BBa_K177026 and received transformant colonies. We also prepared a lot of plates with LB agar with and without antibiotics. After the transformation results we chose TOP10 for subsequent experiments, as the most effective E. coli strain.
Week 2 (2.07-6.07)
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, 10 ul Taq master mix and filled the tubes with nuclease-free water up to 20 ul. Gel electrophoresis of our PCR products showed 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.
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. Thanks to that we could start preparing primers to produce our new parts (through PCR without template) – the 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)
Our new primers finally arrived on our lab tables! We started off with ‘no template PCR’ synthesis of our new promoter and RBSes. We ran electrophoresis of these products, and guess what? Again we were successful! So we ran more ‘no template PCRs’ – this time more RBSes and terminator were synthesized. The next step was clean-up of our PCR products - we checked their concentrations on NanoDrop spectrophotometer. 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)
We ran PCR on the template of products of ligation, to check whether everything went fine. Then we ran gel electrophoresis, which unfortunately showed nothing at all. We tried to repeat all the ligations. We also needed to run another set of PCRs to get some more of our parts. We decided to run standard PCR of our ligation products: e.g promoter+RBS (left primer of promoter and right of RBS)
Week 6 (30.07-3.08)
We checked the concentration of DNA in our ‘no template’ PCR products by spectrophotometry (NanoDrop), some of the concentrations were very satisfactory, some were still pretty low. We found another way to confirm the presence of our parts – digestion using enzymes cutting inside the sequence. Also this time Clone Manager was very helpful and digestion method turned out to be much more useful. Gel electrophoresis of reaction products helped us to confirm wheater ligation went fine.
Week 7 (6.08-10.08)
We cloned out parts on plasmid pJET from CloneJET Cloning Kit, to multiply our parts on a plasmid. 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 – a company sequencing DNA, which is also one of our sponsors. We prepared digestion of RBS, GFP and plasmids pSB1K3 and pSB1C3. Then of course ligation of our parts: RBS+GFP+pSB1C3 and RBS+pSB1K3 and TOP10 transformation. We isolated plasmids from cultures of transformants.
Week 8 (13.08-17.08)
We sent some more samples to sequencing and checked the concentration of the last week PCR products. Thanks to Kaja, we were able to run PCR of pCEP4 – 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 RBSes on pSB1K3 have correct sequence and set some more digestions and ligation of RBSes with pSB1C3, RBSes with pSB1K3, and RBS+GFP+pSB1C3. To check if our RBSes works in E. coli we also ligated them with promoter for this bacterium. Then we ran transformation of TOP10 strain of E.coli with our ligation products. We also 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. Our freezer ran out of competent cells, so we prepared another set of E. coli TOP10 strain. This time we had 90 eppendorf tubes. Unfortunately, our pSB1K3 stock ended - we decided to cut it with XbaI and SpeI of a sample of RBS+pSB1K3, then ran electrophoresis and gelled out empty plasmid. 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. We didn’t want to waste our time so we set digestion and ligation of RBSes+pSB1K3, promoter+pSB1C3, P+RBS+GFP+pSB1K3 and transformate ligation products on TOP10.< br clear="all" />
Week 9 (20.08-24.08)
Finally we could set incubation of Bacillus subtilis 168 cultures. We prepared isolation of our parts on the pJET plasmid and construction of RBS+GFP+pSB1C3 (digestion, ligation, transformation. We also set another PCR on pCEP4 to get oriP. Isolation of plasmids from the previous day and preparing them for sequencing. We also digested pTG262 (EcoRI+PstI) and P+RBS+GFP, ligated and transformated them to TOP10. And... our first transformation of B. subtilis!!! Unfortunately, we weren’t successful in this procedure. Then we continued constructing our parts – promoter + RBS + GFP + pSB1K3 and RBS + LLO + pSB1C3.
Week 10 (27.08-31.08)
Overnight cultures of the transformants resembled growth. None of the transformants with the LLO construct formed colonies – no growth – something is wrong with our construct! We tried repetition of the whole procedure of preparing this construct and transformation.
Next step – reviving BioBricks (mammalian RBSes from the distribution) J63003 and K165002, transformation and inoculation. We prepared overnight cultures for minipreparation of plasmid DNA, after this - digestion of the mammalian RBSes, pSB1C3 and RBS+GFP+pSB1C3. Ligation of mammalian RBSes+RFP+pSB1C3, then transformation and inoculation. Transformants with LLO didn’t grow up. We ran control digestion of the LLO part and electrophoresis to check what’s wrong. We also set PCR for pCEP4 – to get oriP and prepare electrophoresis of the reaction products.
Week 11 (3.09-7.09)
Thank you Edinburgh Team! We received pTG262 plasmid, so we could clone Bacillus constructs we aquired so far. But the first step was E. coli electroporation. After receiving colonies and plasmid isolation, we inoculated B. subtilis 168 to prepare competent cells. We tried to transform them with pUB110 (control) and pTG262.
Great thanks also to Slovenia Team - we got CMV promoter, which we can add to our mammalian construct! This week, we also inoculated SuperFolder GFP construct. PCR problems on pCEP4 template made us design new primers. We digested all previous constructs and preparated a full one – ligation P+RBS+GFP+pSB1C3, subsequently transformation and inoculation. Electrophoresis of digestions proved wrong placements of inserts.
Week 12 (10.09-14.09)
We made isolation of plasmid DNA P+RBS+GFP+pSB1C3, NanoDrop measuring of DNA concentration and prepared samples for sequencing. Then we digested P+RBS+GFP+pSB1C3 construct and ran electrophoresis to check the placement of the insert. PCR of pCEP4 – we set some changes in gradient annealing temperatures. Digestion of all our parts in the purpose of ligation into pSB1A3 and pSB1C3 and transform TOP10 with it.
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!
