End of Summer Summary

Happy end of summer research to all my fellow honors fellows and other summer researchers!

This summer I accomplished a great deal, even though many of my experimentations didn’t yield great result, but that’s chemistry.

Unfortunately, much of the bad results were related to my actual honors thesis. I think I have successfully synthesized the azobenzene alkyne, but I am having a trying time inserting it into GFP. I retested the Azof synthetase that I utilized for my standard azobenzene UAA, and that still worked greatly, but it would not take the new azobenzene UAA. I tried at a standard and lower concentration, because I realized that the UAA itself is toxic at concentrations of 100mM. I performed a synthetase assay on a 96-well plate using six commonly used and promiscuous synthetases at standard and low conentrations of UAA, but none of them showed any promise. When I return to William & Mary next week, I will try a few more synthetase assays, in hopes that I won’t have to perform a synthetase selection (which I’m currently doing for another UAA). I also hope to start synthesizing the next two azobenzene derivatives.

The 1,2,3-triazole cycloaddition (or click) project has been pretty interesting. I have performed organic, protein, and resin clicks using a variety of instruments. I have attempted both catalyst and catalyst-free trials on the microwave and coolmate. The coolmate clicks seem to be working with organic molecules; however, the non-catalyst versions don’t work quite as well on the coolmate as they do on the microwave. Excitingly, I was able to perform protein clicks on the coolmate without completely destroying the protein, as usually happens with the microwave. Unfortunately, while I was able to successfully perform a protein coolmate click, I’ve been unable to repeat the reaction, despite many repeated trials. Perhaps I have been using the wrong protein during these recent trials. Glaser-Hay reactions were tested on the coolmate, and it appears that they do work with a few organic small molecules, and perhaps with protein. On an SDS gel, small bands were detected when protein was utilized; however, optimization still needs to be performed. The catalyst-free organic click project is exciting but the optimization is tedious. I have synthesized one reaction with high yields, but the other reactants I’ve used aren’t as effective. This could be due to the structural differences among the many organic molecules I am testing. Perhaps the catalyst-free click reaction only works with specific types of molecules.

Regarding the selection, the Selenocysteine selection is actually working. We are currently about to prepare for the third round of plating. It was a great struggle to get enough cells for the from the first round, and he had to alter the conditions a little bit to do so. The second round went quite swimmingly so hopefully we are on our way to creating a successful synthetase.

I don’t move back into W&M for a week, so I won’t be able to post for a little why, as it takes quite a bit of work to get back to where I was at during the summer.

I hope everyone is enjoying their last few days of summer, and I can’t wait to see everyone!

Thanks for reading!

-Marshall

Comments

  1. Great work Marshall! I look forward to hearing about the progress on these projects over the year!