Dr. Laura Rowe
Arts and Sciences
Proteins allow daily processes in the cell to occur. A protein consists of amino acids. There are twenty natural amino acids coded for in the DNA of organisms. The natural amino acids can be modified to form unnatural amino acids (UAAs). UAAs have useful characteristics when inserted into a protein of a cell, like the ability of fluoresce, which makes their incorporation important in research. For an UAA to be incorporated into a protein, it must be bound to a transport RNA molecule by an enzyme called aminoacyl tRNA synthetase (aaRS). An existing aaRS was modified in E. Coli bacterial cells to incorporate 3-(2-pyridyl)-L-Alanine since it has metal-binding capabilities. Once incorporated, the UAA acts as a sensor for a metal, making it useful to environmental fields. The aaRS was mutated using saturation mutagenesis at sites L32, V65, W108, G158, A159. The cells were run through a positive screen to determine if the mutated aaRS incorporated the UAA into a green fluorescent protein, which glowed if the UAA was inserted. The results of the positive screen showed mutated aaRSs 2, 4, 7, and 8 incorporated 3-(2-pyridyl)-L-Alanine, while mutated aaRSs 2, 5, 6, 7, 8, and 9 incorporated p-cyanophenylalanine. A negative screen to test if the mutated aaRS only incorporate an UAA, not natural amino acids still present in the cell, will be run on the mutated aaRSs passing the positive screen.
Burkhart, Grace, "Modification of Aminoacyl tRNA Synthetase in Order to Incorporate an Unnatural Amino Acid" (2018). Summer Interdisciplinary Research Symposium. 29.