Team:NRP-UEA-Norwich/Notebook

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Revision as of 21:52, 21 September 2012

NRP UEA iGEM 2012

Welcome to the NRP UEA iGEM 2012 Wiki Lab Book

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

REBECCA AND LUKAS TO WRITE SUMMARY HERE AND ADD PICTURES/GRAPHS ETC. TO LAB BOOK

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 within the 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 to what to do. Everyone had really big ideas as to 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 and find everything ourselves. Needless to say, it took us many weeks to get into the swing of things. As emphasised, things kick started rather slowly.

Initial problems we had 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, 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 potentially, our first BioBricks, we set about planning about looking to ligate reporter proteins to them to both improve the BioBricks and to characterise them. We also set about characterising other PyeaR + GFP.

RFP and CFP BioBricks that we had on our plates, were selected and prepared to be ligated to BM and MB. Possibly due to denaturing, contamination of 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 testing the affects of nitrates on the PyeaR transformed cells, affect of transforming PyeaR, BM and MB 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 to cloning. 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 characterisation off, we continued lab work into week 11, where we were able to use FACs, flow cytometry and a fluorometer. Besides these tools, we also transfected BM an 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.

@@ Line 9: / Line 9: @@
 At the start of the 10 weeks, we were a little lost as to what to do. Everyone had really big ideas as to 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 and find everything ourselves. Needless to say, it took us many weeks to get into the swing of things. As emphasised, things kick started rather slowly.
-Initial problems we had 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, 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. The constructs were also ready to be sent off for synthesis.
+Initial problems we had 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, 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 potentially, our first BioBricks, we set about planning about looking to ligate reporter proteins to them to both improve the BioBricks and to characterise them. RFP and CFP BioBricks that we had on our plates, were selected and prepared to be ligated to BM and MB. This process began in week
+Onto the latter half of the project, we still had much to do. With potentially, our first BioBricks, we set about planning about looking to ligate reporter proteins to them to both improve the BioBricks and to characterise them. We also set about characterising other PyeaR + GFP.
+RFP and CFP BioBricks that we had on our plates, were selected and prepared to be ligated to BM and MB. Possibly due to denaturing, contamination of 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 testing the affects of nitrates on the PyeaR transformed cells, affect of transforming PyeaR, BM and MB 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 to cloning. 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 characterisation off, we continued lab work into week 11, where we were able to use FACs, flow cytometry and a fluorometer. Besides these tools, we also transfected BM an 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.