Presented by Alissa Meo
Meredith College CSA Day 2020
#MeredithCSA
View the 2020 CSA Program Booklet at [ Ссылка ]
Abstract:
Violacein, a purple-pigmented protein commonly found in the bacterium Chromobacterium violaceum, is regulated by quorum sensing mechanisms, and functions as a broad-spectrum antibiotic, antifungal, and powerful antioxidant. The genes that code for violacein are found in the vioABCDE operon and span over 10kb. The vioA gene codes for a non-pigmented precursor to violacein called flavin-dependent L-tryptophan oxidase. Any disruptions or mutations in the vioA gene will cease the production of violacein completely or produce non-functional violacein. The ultimate goal of the research was to induce an overexpression of the VioA protein in Escherichia coli for purification. VioA is encoded by the vioA (approximately 1.25 kb long) in C. violaceum, the first gene in the violacein operon. The vioA gene was isolated by methods of polymerase chain reaction (PCR). Primers for the PCR were designed using the Primer3 online primer design tool to amplify the entire coding sequence of the vioA gene, and extracted genomic DNA from C. violaceum was used as a template. This PCR product was cloned into the TA cloning vector, pGEM-T Easy plasmid, and transformed into a methylation negative E. coli strain and grown on ampicillin selection plates. Ultimately, this gene will be subcloned into the expression vector, PinPoint Xa-1, which will enable purification of the VioA protein.
