Jun
25

An immediate success, followed by several roadblocks

As research has progressed so far in the summer, it is becoming increasingly clear how even simple tasks can become challenges of their own. In the first couple of weeks I was able to focus in on one location of our mutation in the protein, which allows me to concentrate all my efforts on expressing one or two DNA plasmids rather than going between several different versions. This has increased my troubleshooting productivity greatly, as I can focus on fixing issues in the protocol rather than wondering if it’s an issue with the plasmid or protein itself being more fickle. I was able to express my protein with an unnatural amino acid (UAA) in a conjugated protein that promotes stability. I then utilized a chemical reaction to attach a fluorophore to the UAA, which was successful. In conjunction with the Kerscher lab, we proceeded with a binding assay on beads and a resin. While the resin did bind, the protein concentration was not high enough to determine conclusively if the bead binding was successful or not. I attempted to express a larger volume of my protein in order to increase the concentration of protein so we could proceed with more regular binding assays and fluorescence reactions, but unfortunately after several attempts and a couple of weeks I did not succeed in increasing the scale of protein expression. I have since scaled back the expression again, and it appears that protein is present. I am currently carrying out a fluorescence reaction in order to, hopefully, proceed with refining the binding assays. Biological research is particularly daunting in particular, as at any moment one of my multiple multi-day experiments may simply decide that it is too hot, or too cold, or too concentrated, or too dilute, and the cells will die. It is then my job to decide what happened, and determine how to fix the issue to ensure that it does not happen again. When my experiments do work, however, it is very encouraging. I am always surprised how much motivation and drive to succeed I am able to pull from the slightest whiff of success, spurring me on to try all the more.BLR 5-3-18 MBP 341 #2 and 6 GH Coomassie (John) BLR 6-11-18 MBP 341 amylose resin and SUMO bead coomassie BLR 6-13-18 MBP 341 large expressions

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