Iterative Site-Directed Mutagenesis Towards the Directed Evolution of Enzymes, Blog post #1

The topic Arianna, Dr. Chang and I are researching is Iterative Site-Directed Mutagenesis Towards the Directed Evolution of Enzymes. The purpose of this project is to understand the self-assembling properties of silicateins and try to develop new forms of cathepsin-L which will adopt these properties. To accomplish this, we must determine which amino acids residues are responsible for the self-assembling properties of silicateins. Once we have this knowledge, we will make changes to different amino acids within cathepsin-L. Ultimately, the goal of this research project is to aid future studies in utilizing the self-assembling cathepsin-L to create materials for the use in medical implants and optical materials.

So far, our time spent in the lab has been focused on preliminary preparation of materials such as antibiotic containing plates, chemical transformations with e. coli, and mini-preps towards DNA analysis. We have also spent some time conducting a literature search using Web of Knowledge. We researched relevant literature published in recent years that could help us throughout our research project. During this research, Arianna and I will learn techniques in protein mutagenesis as well as how to characterize hydrophobic and electrostatic protein-protein interactions. The methods we will be using in the future to answer our research questions include: expressing, purifying, manipulating and characterizing proteins; forming and refining silica mineralization assays, both in the absence and presence of proteins; completing scanning and transmission electron microscopy, atomic force microscopy and focus-ion beam milling; and finally, analyzing our complex datasets in order to accurately interpret the results.

This is my first time working in a research lab so each step along the way has been a new opportunity for me to learn. From the first day learning how to use the autoclave, to our last meeting when we successfully transformed plasmid containing the cg-LDH gene into a host organism, I have learned a wealth of new lab skills. Exposing myself to new academic experiences and growing as a student as well as a team member are the key things I would like to achieve from this project. Through our research, I hope to expand my knowledge of proteins, enzymes, and the amino acids which are the building blocks behind them.


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