Reflecting on Spatial Thinking

As the Summer draws to a close, I am filled with the nostalgic feelings that accompany returning to school. This summer 5 AidData Summer Fellows traveled to the Philippines and incredible 70 day experience.

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The end, for now

I have been working steadily over the summer to translate, score, and analyze my results for the project. I have also met with Professor Aday a few times to discuss the work and its implications. Overall, we have decided that this work should continue and I am going to pursue a few conferences and publishing the final paper. Which is very exciting! I certainly didn’t think that was possible, but I am glad we have been able to get the project so far so soon!

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Done in Andahuaylas, Off to Lima!

After I finished my work with the kids, I also finished with the adults. The last two classes went pretty smoothly with them. There were a lot of questions and a lot of new ideas, but I think they were generally receptive.

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Looking forward…

I was really happy with the protein I was able to synthesize, the experiments I was able to conduct and the results I received! By analyzing these results, I have been able to come up with new ideas for future experiments we can conduct by incorporating different UAAs into GFP. Continuing with the 3FY work done this summer, I would like to incorporate other fluortyrosines including di- and tri- fluorotyrosines. I would conduct similar titration experiments to see if the pKa shifts through the addition of fluorines. Finally, I will conduct similar fluorescence spectral analysis on the ortho-nitrobenzyl tyrosine GFP as well.

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3FY Titration Results

Excited by the successful results of the p-propargyloxyphenylalanine GFP Glaser-Hay fluorescence results, I moved on to studying the 3-fluorotyrosine (3FY) GFP I had synthesized earlier. This was a project that had be started earlier by a student who had graduated this past semester so I was thrilled to take it on. 3-fluorotyrosine is another amino acid that excites me as the fluorine groups can significantly modify the chemical properties of the protein. In particular, I decided to look at the effects of 3FY on the pKa of the protein. This was done by conducting titration experiments on the 3FY-GFP and wild-type (WT) GFP.  Titration experiments involve adding an acid or a base to protonate or deprotonate a compound. Emission spectra can tell us about the amount of protein that is protonated or deprotonated as the two forms emit different wavelengths of light. The protonated peak for 3FY-GFP occurs around 450 nm while the protonated peak is at around 525 nm. By calculating the ratio of the protonated peak to the deprotonated peak, we can determine what percentage of the protein is protonated and deprotonated. A baseline spectra was acquired for both 3FY-GFP and wild-type GFP. Subsequently, acid or base was added to the sample in small intervals followed by another emission reading. This was repeated several times to achieve a full titration curve. As acid is added to the sample the ratio of the protonated to deprotonated peaks went up while the reverse was true for base additions. We observed significant changes in the pKa of the 3FY as compared to WT-GP when the titration curves were plotted.

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