Another Update Part 1: Failure

Hello all!

I know I have been absent for a while, but I can assure you that I’ve been quite busy with research, and especially with busy applying to grad school! Because I have a plethora to discuss, I will make separate posts.

This post will concern all of the unnatural amino acid projects I have been working on.

The first is the azobenzene derivative project. After synthesizing the alkyne derivative, I ran into many roadblocks, namely my inability to incorporate it into GFP. To do this, we usually use a synthetase specifically designed for the UAA itself, and for the regular azobenzene we had a synthetase, but it has not worked for the derivative. Instead I performed a synthetase assay with about a dozen different synthetases. This required transforming competent bacteria with two separate plasmids, growing them, and inducing them on a 96-well plate. Unfortunately, the fruits of my labor were sour as I didn’t find a single synthetase that worked. I then attempted to lower the concentration of the UAA, theorizing that the derivative itself was cytotoxic, but unfortunately that did not help. I have since tabled the project to focus on more pressing research, as well as graduate applications, which always find a way to eat up my time. Make no mistake, I will return to this project, hopefully with greater optimism.

The selenocysteine project has also seen a few….difficulties. We had been in progress with selecting a synthetase, a much different process than above. In this case, we utilize a DNA/synthetase library of billions of different synthetases, and select for one that can use our UAA by a process of positive and negative selections, using various antibiotics and agar. We got through the first positive and the first negative rounds but got to a road block. While I was selecting for azobenzene, I tried using the same synthetases for selenocysteine, and I actually got a hit. We tried to express protein, but the concentrations seemed low. We are currently trying a variety of expression and UAA concentrations to find the conditions for optimum protein production. Hopefully we will see good results soon!

Thanks for reading part one, part two will be up soon.