The purpose of my project was to express and measure in vivo activity of the TrpM enzyme in E. coli. TrpM is part of a methylase family of enzymes that adds a methyl group to a base molecule, in the case of TrpM, the base molecule is the aromatic amino acid tryptophan. To do this I had to learn many new laboratory procedures such as DNA purification, gel electrophoresis, microbial fermentation, and HPLC analysis. Each of these procedures were used throughout the steps of my project. First, I cloned the TrpM gene and inserted it into two different plasmids, pFB06, which had a resistance to kanamycin and pETM6, which had a resistance to ampicillin. Each of these antibiotic resistant plasmids were then introduced to the E. coli strain, BL21 starTM (DE3). After the bacteria had been grown and induced with IPTG, they were tested under different conditions, including: varied induction timing, induction vs. no induction, media containing tryptophan vs. without tryptophan, and two different production strains containing pFB06 and pETM6. To analyze the success of our experiments, we prepared samples of each culture for analysis by HPLC. The results showed significant variance in the HPLC spectra between sample replicates, however, there were multiple new peaks of interest that give possible evidence that TrpM successfully methylated tryptophan in our culture broth . I plan to conduct additional trials next fall to improve my fermentation technique, gain more consistent research findings, and further strengthen my experimental results.
Author: Fiona Kanis
Faculty Advisor: Dr. J Andrew Jones, Department of Chemical, Paper, and Biomedical Engineering
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