MARCH - JULY
Planning! We spent a long time umming and ahhing about potential projects. We only decided to put all our efforts into one just a week before we were due to start in the lab at the beginning of July!
Discussing options for CRISPRi and transposons to give assortment of levels for products. We spent a lot of time in meetings and planning to make sure that we were sure about our project. We went bowling as a team this week and discovered that Natalia is an incredibly precise bowler... hopefully her precision transfers to her pipetting skills - all will be revealed later.
This week we really got our heads down and the biochemists planned our constructs and designed primers. In the meantime the rest of the team worked on outreach (i.e. sending hundreds of e-mails!). Vikram, the computer scientist, worked on lots of complex computer stuff and worked endlessly on our game, a Portal mod. Jake made us a new logo and designed a front page for our wiki which looks super professional now.
This week we started lab work! 5 of us (biotechnologists and biochemists) were in the lab learning the techniques needed for iGEM.
Monday - Primers and enzymes were ordered for one of our two constructs today so we started on the InterLab study until they arrive! We transformed the controls and devices into DH5a competent cells that were provided for us. We set up overnight cultures of TOP10 cells for making electrocompetent cells, and we also made O/N cultures of E.coli carrying the backbone we need for our pReporter which was donated by a lab member. This is called pSTLS. Vikram released a teaser trailer for the game we have made (INSERT LINK HERE). Vik's computer then died
Tuesday - Vik's computer is alive again. Unfortunately our transformations only gave very small colonies on some plates, and none on others. We re-streaked those colonies that we did have. We extracted plasmid DNA from the O/N cultures of pSTLS and others for the lab to get some practice. We learnt to use the NanoDrop to quantify DNA. Outside of the lab, Georgette was busy creating our very first vlog which will be shared on the social media pages. Vik is looking at building hardware. Today we also skyped Edinburgh UG team to discuss possible collaborations
Wednesday - Waiting for primers to arrive. NEB sent us some enzymes and competent cells so thanks to NEB!
Thursday - Today we learnt to electroporate DH5a and TOP10 cells. We transformed them with either a plasmid containing dCas9 or our low copy backbone, pSTLS. We also set up overnight cultures for the InterLab Study.
Friday - DISASTER! Two of our overnight colonies didn't grow so we have to restart the overnights on Monday. Ah well, we are learning what science is like in reality. We went for a social today - Laser quest! Georgette won by far, Ellie lost miserably (-8300 points). Georgette was appointed new team leader due to Ellie's shocking performance... just kidding!
This week we carried on with the InterLab study and waited for our primers to be delivered.
Monday - we re-set up overnight cultures for the InterLab study today.
Tuesday - all our cultures grew so we proceeded with InterLab GFP measurements... they weren't what we were expecting! Jake and Vik skyped Bristol iGEM to see whether we can help them improve the wiki design process.
Wednesday - we analysed the data from the InterLab study today and e-mailed some important people for outreach purposes.
Thursday - Jake changed the nav bar today. Chris made one of our many games.
Friday - we took team photos, ate fish and chips and OUR PRIMERS ARRIVED! This means we can properly start in the lab on Monday!!
Monday - today the primers arrived so we had a long day in the lab doing 46 PCR reactions! We learnt how to set up PCR and load agarose gels, and how interesting waiting around for PCR is. We got lots of interesting results. Our original PCRs were using 1ng template and touchdown from 70-60oC for 10 cycles followed by 25 at 65oC. These conditions were great for getting most of the components to amplify, however we had to optimise a few. For example the dCas9 and low copy backbone didn't amplify first time around, so we used a new strategy - using various amounts of template with duplicates placed at different annealing temperatures. One was placed in a touchdown setting as before but with annealing temperatures dropping from 60 to 50oC followed by 25 cycles at 65oC. The other was placed at 55oC for all cycles. We also didn't get good amplification of P1, P2, P3, P4, T1 or T2 so we repeated their PCR using touchdown from 70 to 60 as before but with 5 more cycles.
Tuesday - we checked the optimised PCR results today. We managed to get P1, P2, P3, P4 amplified, and T1 and T2 amplified but there were far too many bands to be certain we were amplifying the right thing. So we re-amplified T1 and T2 using PCR with touchdown 60-50 as above. T1 was successfully amplified using this approach, however we got really strange bands for T2 so we went back to the samples from the previous PCR to see whether we could use those instead for T2. Our optimised PCR showed dCas9 was successfully amplified in all conditions - yay! The low copy backbone only amplified at 55oC using 1ng template so we repeated this PCR using those settings to generate enough DNA for later. This PCR was successful.
Wednesday - now that we had most of the components, we could digest them. P1-5 + PE, the strong and weak RBS linked GFPs, and T2 were digested with BsaI. For the gRNA plasmid, J23119, gRNA 1-5 and gRNA0 as well as Tfdx were digested with BsaI. We digested 1ug for all of the components except the GFPs. 2ug of both backbones were digested too. The gRNA backbone was digested with SalI and AscI, whereas the low copy backbone was digested with SalI and BsteII. For the dCas9 brick, we digested dCas9 with Bsa1, T1 with BsaI and SalI, and PdCas9 with BsteII and BsaI. We PCR purified all of the digested before ligating together the bricks using T4 DNA ligase. We had problems digesting gRNA 4 as the DNA concentration dropped to 2ng/ul on two separate occasions, so the whole process was repeated for gRNA 4. 50ng of each brick was used for the brick ligations whereas 100ng of the backbones were used. 13.3ng of T1 was used and 7.2ng of PdCas9. Ligations were carried out for 2.5 hours.</li>
Thursday - today we checked whether the ligations had worked. We tried to amplify using our brick forward and reverse primers for all bricks, however the results showed amplification of products much smaller than expected. We suspected a few problems so took a while to look through the primers using SnapGene and identified a few tweaks that could be made to the gRNA brick primers, so we ordered new primers for that. We were very confused by the promoter-GFP-terminator bricks as the products should be over 1kb whereas they were showing up at under 500bp. We decided to troubleshoot the PCR by using the brick forward primer and the sGFP reverse primer on P1, P2, P3, PE on the corresponding ligations. Today we also decided to ligate the sgRNAs with their promoter, terminator and the backbone in a 4 fragment ligation using T4 ligase overnight at room temperature.</li>
Friday - another late finish today! Today we checked whether the PCRs worked with the brick forward primers and the sGFP reverse primer for P2, P3, P4 and PE "bricks" using our ligations as templates. They did! So that gave us hope that the promoter and sGFP are attached properly. We realised there was a problem with T2 amplification so we decided to switch to using T3 instead. So we created primer dimers of T3 and amplified it before PCR purification, ready for digestion on Monday. We noticed another problem with P1 which meant that it would not amplify using the primers we currently had, we made larger quantities of PCR of P2-5 + PE bricks using the brick forward primer and either strong and weakGFP reverse primers. Unfortunately the gel showed that the wGFP bricks did not amplify but the sGFP bricks did so we are a bit confused. We cut out the bands which had worked ready for gel extraction on Monday. Today we also dialysed our ligations which had been running overnight, and transformed these into TOP10 by electroporation and plated them on chloramphenicol plates to select for transformants. We also checked the transformation plates from yesterday which hopefully had the low copy backbone plus dCas9, PdCas9 and T1 in - at the end of Friday there were 2 colonies from being left in 30 degrees for around 24 hours - good news!