Wednesday, July 8, 2009

Building Circuits: A Few Successful Moves to the End, but too many Failures...

Hi! You heard from me last week in my third-person commentary and now I’m back in the first-person flesh. My name is Vicki and I recently graduated from the Engineering Science program at the University of Toronto, with a major in biomedical engineering. I grew up in Calgary and am very excited to help my hometown kick some synthetic biology butt. When I am not indulging in the satisfaction that only a properly-sequenced synthetic BioBricked circuit can provide, I am usually either swimming, skating, biking, learning German or a combination of any of the above (ever try riding a bicycle whilst on skates? It’s as perilous as it sounds!).

Over the last few weeks, I have been deeply entrenched in converting a mutant protein sequence in a TOPO plasmid into a fully functional and biobricked sequence in a pSB1AK3 plasmid, complete with the appropriate and properly-integrated promoter, RBS and terminator sequences. Indeed, it has been a most gruelling month of gradient PCRs, colony PCRs, plasmid isolations, restriction digests, ligations and – craziest of all – understanding what I’m doing and explaining it to the lab group in coherent sentences! Because even though the principles of synthetic biology and biobricking are supposed to make genetic engineering so easy that even an engineer like me can work with it, it’s really quite challenging when you cannot see what is really happening in terms of molecular interactions on the nano-scale and smaller. Although many parts of the project have flummoxed me, I am developing a better appreciation of what I am doing as I gain more experience in the lab.

So, here is my work to date.

Battle 1: Vicki vs gradient PCR of LuxOD47A in TOPO. The purpose of this step was to use biobrick gene-specific primers to make copies of a biobricked version of LuxOD47A.

Winner: Vicki

Battle 2: Vicki vs ligation of LuxOD47A into the pSB1AC3 plasmid. This is so that the gene sequence can be integrated into competent TOP 10 bacteria. Prefix-end cuts were made with EcoRI and XbaI (in separate samples).

Winner (VK vs EcoRI cuts): EcoRI cuts L. The restriction digests of these look like someone painted white-out in the 12 kb range just to spite me.

Winner (VK vs XbaI cuts): Vicki. The EcoRI enzyme sample that wasn’t up to par is spending its days in solitary confinement at room temperature. Indeed, things are so much cleaner when enzymes decide to cooperate.

Battle 3: Vicki vs the sequencing machine, part I. After a successful restriction digest of the XbaI-cut samples, I sent them down for confirmation that my PCR and restriction digest results weren’t just lying to me.

Winner: Vicki

Battle 4: Vicki vs integration of promoter and terminator sequences, attempt I. I tried both to see which would work best.

Winner (VK vs J13002 promoter): J13002 promoter L. That one didn’t integrate in any of the colonies that I PCR’d and RD’d

Winner (VK vs B0015 terminator): Vicki. We’ll move forward with a newly-made LuxOD47A-B0015 construct, while the attempted J13002-LuxOD47A samples can go roast in the autoclave.

Battle 5: Vicki vs the sequencing machine, part II. Yea, I know I skipped a lot of steps here. It wouldn’t tell you anything interesting beyond what you’ve already read. After good restriction digests and colony PCR results that would have been fine if not for a superfluous negative control band that didn’t show up anywhere else, we sequenced anyway. We had a scare when a simple [Crtl+F _ (B0015 sequence)] didn’t yield any results on the text file, but were reassured when we blasted the sequences against each other as that algorithm accounts for the reverse sequence given by the reverse primers that I used.

Winner: Vicki, with credit to BLAST

Battle 6: Vicki vs the integration of the nefarious promoter sequence. Lest the J13002 samples go the same way as our uncooperative EcoRI enzyme, we decided to attempt this integration thing one last time. Two colonies behaved appropriately, and the J-part lived to see another day.

Winner: Vicki

Battle 7: Vicki vs the sequencing machine, part III. Although I had to wait four days for my results, they were what we expected.

Winner: Vicki

This takes us to now. I need to make more of the biobricked sequence in question by letting it grow in bacteria, which I’ll do later today so that it can grow overnight. Results to come!

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