Showcase of Undergraduate Research and Creative Endeavors (SOURCE)

Investigation and Characterization of Mycobacteriophage ExplosioNervosa

Bacteriophages are viruses that replicate using a bacterial host, which has resulted in a dynamic co-evolutionary relationship. To further our understanding of this relationship, SEA-GENES students have been investigating the mycobacteriophage ExplosioNervosa. This phage was found and isolated on Winthrop University’s campus in 2017. ExplosioNervosa is a cluster A9 bacteriophage with a Siphoviridae morphology and has 96 predicted genes. Of these genes, only 36 are predicted to have a known function. Since the majority of ExplosioNervosa’s genes have no known function, it is important to further identify the characteristics of each gene. Throughout the semester, students were individually assigned a set of genes to isolate and further characterize. Using molecular cloning techniques, students isolated and amplified each of ExplosioNervosa’s genes. Utilizing PCR, each gene was exponentially amplified. Gel electrophoresis was used to verify that the correct amplicon lengths were generated. The gene amplicons were then isothermally assembled into pExTra plasmids. Once assembled with the gene insert, pExTra plasmids were transformed into Escherichia coli to replicate and generate plasmid DNA. The plasmid DNA was then verified for the presence of each cloned gene. The next step involves transforming the pExTra plasmids + gene insert into bacterial host Mycobacterium smegmatis using electroporation. Cytotoxicity assays can then be performed to determine what effect gene expression has on the growth of its host, M. smegmatis. This phenotypic assay allows for phage-host interactions to be observed and is an initial step to further characterize gene function.