Showcase of Undergraduate Research and Creative Endeavors (SOURCE)

Characterization and Analysis of Mycobacteriophage Cain’s Genes

The majority of bacteriophage genes have no predicted function, which provides a wealth of opportunity for investigation, characterization and potential downstream applications. The work described here includes, and extends, the research initiated by Winthrop University students as part of the SEA (Science Education Alliance) GENES (Gene-function Exploration by a Network of Emerging Scientists) program, sponsored by the Howard Hughes Medical Institute (HHMI). Our phage of interest is a K6 sub-cluster mycobacteriophage; Cain, which has a genome consisting of 100 diverse genes. To gain clues to their functions, the workflow began by cloning each of Cain’s genes individually using a shuttle vector (pExTra), and amplifying in Escherichia coli. Each pExTra + gene was then transformed into its bacterial host Mycobacterium smegmatis. A phenotypic assay then enabled the observation of host growth inhibition after inducing the expression of each phage gene individually. As a result of our work, 18 of Cain’s genes have demonstrated this cytotoxic trait which infers some sort of interaction with the host’s proteome. All cloned genes were concurrently sequence verified by comparing against the published Cain genome using in silico NCBI sequence alignment tools and SnapGene Viewer software. Our group is also performing Bacterial-2-Hybrid (B2H) assays to investigate the interactions occurring between a phage gene product and its host’s proteome, in more detail. Elucidating specific interactions at the phage-host interface may provide opportunities to exploit previously unknown proteins or manipulate the outcome of the microbial co-existence.