Team:NRP-UEA-Norwich/Notebook

From 2012.igem.org

Welcome to the NRP UEA iGEM 2012 Wiki Lab Book

Please choose the relevant link to access our diary of that week!

Week Zero | Week One | Week Two | Week Three | Week Four | Week Five | Week Six | Week Seven | Week Eight | Week Nine | Week Ten | Week Eleven | Lab Protocols | Experiments

The NRP-UEA team was funded by the Wellcome trust for 10 weeks commencing from July 9th to September 14th. Leading up to the start day, the team met up to discuss the scope, the runnings and the time frame within which we had to complete a synthetic biology project. It was during that month leading up to the start date that we decided to build a sensor for nitric oxide and the rest was history, or at least slowly fell into place.

At the start of the 10 weeks, we were a little lost as what to do. Everyone had really big ideas as how to carry out the project but relatively little experience in research. Having been a little molly coddled in lab practice, we were suddenly doing everything ourselves, finding solutions and using research to solve problems. Needless to say, it took us many weeks to get into the swing of things. As emphasised, things kick started rather slowly.

Initial problems included transforming iGEM BioBricks, DNA isolation, gel purification and ligations. In general we encountered many challenges in all steps of cloning. However, with jokes and encouragement from each other, we worked on the finicky details of the protocols, such as a flick here, a centrifuge short spin there and lower antibiotic resistance there. Slowly but steadily we improved our lab skills. Around week 4 to 5, lab work began to fall piece by piece into place. It was also around this time that our grand ideas and designs for building a quantitative and specific modular sensor came together. All in all by the half way point, things were looking up. We had successfully cloned BM and MB into the pSB1C3 iGEM backbone and isolated the DNA. We had prepared reporter proteins: RFP (BBa_R0080) and eCFP (BBa_E0420) for ligation with the potential BioBricks. The constructs were also ready to be sent off for synthesis.

Onto the latter half of the project, we still had much to do. With our potential first BioBricks, we set about planning to ligate reporter proteins to them to both improve the BioBricks and characterise them. We also set started to further characterising other reporter such as PyeaR + GFP.

RFP and CFP BioBricks that we had transformed, were selected and prepared to be ligated to BM and MB. Possibly due to denaturing, contamination and mutation, the eCFP and RFP DNA we had hydrated was not transforming and so we decided to use other BioBricks. However none of the ones on our plates had ribosome binding sites and confirmed sequences. Therefore, we streaked the DNA we had from the original transformation plates of RFP and CFP and restarted the cloning process. Towards the end of the allotted 10 weeks, we finally cloned the reporter proteins into our BioBricks and characterised them.

The growth study characterisations we carried out involved measuring the affects of nitrates on PyeaR + GFP transformed cells and the affect PyeaR + GFP, BM and MB Biobricks on the growth rate of E. coli. The results of these were at times what we expected and at times not.

In week 9, the synthesised DNA of our constructs arrived! Despite this being later than we originally had hoped for, we were determined to make things work and immediately set off to get started on the cloning process. By this point, everyone was very rehearsed in the methods of cloning and almost everything worked first time. It was also through the cloning of our constructs that new champions of miniprepping arose. By the end of week 10, we had successfully cloned Construct 1 and 2 into pSB1C3 and sent the DNA off for sequencing and to iGEM.

To finish the characterisation process off, we continued lab work into week 11, where we were able to use FACs, flow cytometry and a fluorometer to gain insight of the functionality of the new Biobricks. Besides these tools, we also transfected BM and MB into mammalian cells, prooving that the combination of mammalian and bacterial promoters brought about flexibility of chassis. Using these, we were able to fully characterise BM and MB.

For full details of the lab work carried out, check out the individual weeks. The details of the protocols used are stated and also the full details of experiments and results can be found in the experiments page.