Summer Research 2015: Second Update!

Good Evening

We are halfway down with summer research, and while the progress I’ve made is exciting, the fact that we are closer to the end of the summer is frightening as I still have much to do!

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Summer Research 2015: First Update!

Howdy,

The first few weeks of research have gone by fairly quickly, but I’m happy to report great progress! Since I work full time, I have been assigned several projects that I work on throughout the week, let me quickly go over them.

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Abstract: Unnatural Amino Acid Azobenzene Derivatives

Hello!

My name is Marshall Padilla and I am soon to be a rising Senior Chemistry Major. I am back again this summer to continue my azobenzene work through an Honors Fellowship. I would first like to thank the Chemistry Department here at William & Mary for granting me the Llanso-Sherman scholarship, which will fund my Honors research this summer. Last summer I synthesized azobenzene, an unnatural amino acid (UAA) that has an azobenzene R group. I inserted it into Green Fluorescent Protein (GFP) at several different spots and demonstrated that by utilizing azobenzene’s photoisomerability, I could alter the fluorescence of GFP reversibly- meaning I could shift the fluorescence of GFP and reconvert it back. This was shown both in pure protein extract and in vivo, as bacteria were grown that were made able to synthesize of GFP, and demonstrate fluorescence reversibility due to azobenzene.

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Summer Summary

While the summer research has come to an end, my passion and eagerness for more research has only been enhanced.

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Third Post!

While my time in the Young Lab has come to a close, I still have much to discuss. In the last few weeks I started on an actual experiment for the AzoBen UAA. I took agar plates, which are plates that bacteria grow on, and created an environment where bacteria can express the photoswitchable nature of GFP with my UAA. I took four plates, and to three of them I added 10 ml of agar, 10  μl of IPTG, 10  μl of Arabinose, 10  μl of chloramphenicol, 10 μl of Ampicillin, and 100 μl of AzoBen. On the fourth plate, I repeated the same procedure, except I did not add AzoBen. I then plated bacteria that could inherently grow GFP. The purpose of this setup, was to test whether I could detect changes in fluorescence when I irradiated the bacteria. Since three of the plates contained GFP with AzoBen, they theoretically should be able to change the fluorescence. I took one plate, irradiated half of it for 10 minutes, at 365nm. I then left the plate overnight in an incubator. When I came back and checked the fluorescence, I noticed that both sides were the same. This meant that either the experiment did not work, or that the plate did change, but changed back. Hoping that the result meant the latter, I took one plate and irradiated half of it for 10 minutes at 365nm, and checked it after an hour. To my joy, one side had a different fluorescence reading than the other. This meant that the experiment had work; but now I needed to check if the bacteria reverted back. After a few hours of hopeful checking, I saw that the irradiated bacteria were changing back to their original fluorescence! This was fantastic news, as I now have supporting evidence that my UAA is able to alter the function of GFP, but is also able to undo these changes.

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