Iterative Site-Directed Mutagenesis Towards the Directed Evolution of Cathepsin-L Post 2

 

This project has ignited my interest in scientific research and I will continue this project, and another one that will focus on the isolation, construction and modification of a specific yeast killer toxin, K28. I have gained key laboratory skills and protocols that I can utilize in my future experiments and classes. The opportunity to truly experience first-hand the molecular biochemistry reactions was enormously valuable. Many students only hear and learn about some of the processes that I was able to perform in the lab. An example of such was affinity chromatography, a procedure that can be followed in order to separate different biochemical molecules from a mixture based on an extremely specific interaction between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid. This was something that I learned about in my recent cellular and molecular biology class. In addition to this, I also became more experienced in scientific poster creation and presentation. I assisted my group and instructor with putting together a scientific poster that was presented at the 2018 Gordon Research Conference on Biomineralization. This is an exceptionally useful tool to possess because in my career as a science major, I will need to be able to effectively create and present scientific posters, whether they are for conferences or classes.

As is the case in science, obstacles and difficulties were to be expected. We did have trouble initially because the first construct utilized was not useful to express our protein of interest. This construct was the  pUC57 vector that contained the cgLDH gene, which was purchased from GeneWiz, and DH5ɑ E. coli cells, purchased from New England Biolabs. While the vector was effectively compatible with the DH5ɑ E. coli cells, this was ultimately not a good system for producing and isolating the protein of interest. Because of this issue, we were initially not able to isolate cgLDH protein during affinity chromatography, which raised many questions. During our second attempt, we used a pET11a vector with the cgLDH gene, which was purchased from GeneScript, and T7 express E. coli cells, which were purchased from New England Biolabs. This new setup produced the cgLDH protein on the first attempt, however when we checked the approximate concentration using a spectrophotometer, we noticed that we only produced a very small amount. In the future, we can optimize the expression conditions in order to increase the amount of cgLDH produced. Now that we have an established system that works, we can apply this to research with cathepsin-L and see if this protein can be expressed using the T7 E. coli cells and the pET11a vector. If so, we can additionally optimize the growth conditions to produce this protein.

 

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