Grad Diary 2/15/10

As often happens in science, my project has taken a bit of a turn recently. Up till now, I have been characterizing the properties of a GFP-E2 fusion protein that would allow for visualization of viral episomes in vivo (since E2 directly binds to the viral episome and regulates replication and transcription). In vitro analyses of binding and gene transcriptional activation of the two GFP fusion proteins I had showed that they both, to differing degrees, did seem to bind and act upon their target sequences. However, when I tried transfecting these constructs into cell lines which already harbor HPV episomes, expression of the proteins was very low and did not exhibit the correct phenotype. I could spend a lot more time making new GFP mutants with perhaps a longer linker region or whatever, but Jian and I think that would be a lot of work for not very much payoff, so we are switching gears. The upshot is that I have become a lot more comfortable with cell culture, I learned three separate transfection techniques, and I reviewed how to western blot and fix and stain cells for fluorescent microscopy.

The new approach involves using an old friend in the molecular biology world, the lac operon. This "operon" consists of three genes and regulatory elements that feed into lactose metabolism when glucose reserves are scarce; it is present in both prokaryotes and eukaryotes.

The lac operator sequence is bound by LacI (inducer) rather tightly, so our strategy is to clone into the HPV 16 genome a certain number of repeats of the LacO sequence to create a binding site for flourescently labeled LacI protein. This has the advantage over the previous system in that neither LacO or LacI would be involved in the regulation of transcription/replication, so tagging the episome shouldn't adversely effect these processes (whereas GFP-E2 might not function as well in vivo as a regulator).

This is going to involve a whole lot of molecular biology work (restrction digests, ligations, a lot of gel electrophoresis and DNA purification) but fortunately I am already pretty comfortable with most of these techniques. It's going to take a bit of time but I feel pretty good that I will be able to get reasonable progress before the end of my rotation.