Showcase of Undergraduate Research and Creative Endeavors (SOURCE)

Introduction of the Bacterial-2-Hybrid Assay into the SEA-GENES workflow.

Increasing our understanding of bacteriophage-host interactions is crucial, especially as advances in the use of phages therapeutically looks likely. As a part of the SEA (Science Education Alliance) GENES (Gene-function Exploration by Network of Emerging Scientists) program, we have identified 18 genes in Mycobacterium phage Cain that cause the inhibition of growth of its bacterial host; Mycobacterium smegmatis. In order to explore this interactive effect further, a Bacterial-2-Hybrid (B2H) assay has been implemented to help characterize protein-protein interaction (PPIs) between the phage and host’s expressed genes. Each of the previously cloned phage genes were transferred into a vector; p2Hα. The p2Hα + gene was then transformed into a specially engineered strain ofEscherichia coli; B2H SELECT. This cell line contains a library of random fragments ofthe M. smegmatisgenome, housed in another plasmid; pCI. Following induction, both our phage gene of interest and the M. smegmatis gene fragments, are expressed as independent fusion proteins. Phenotypic indicators monitor pairwise interactions between the proteins using an engineered test promoter, reporters and genetic selectors. In this way we can assess PPIs between an individual phage gene product and up to a million different host cell protein fragments. pCI plasmids are sequenced to reveal the host’s gene product(s) that is associating with our phage gene product. Consequently, the interactive partnerships between a bacterial host and its viral parasite reveals clues to phage gene function, and may lead to a number of beneficial applications in science.