The Ko lab studies how genotypic variations contribute to the severity of infectious diseases. By infecting almost a thousand different cell lines, the lab has identified a single-nucleotide polymorphism (SNP) associated with variations in infection phenotypes by Yersinia pestis, the causative agent of the bubonic plague. The SNP is on a gene that codes for a transmembrane immunoreceptor. My mentor Rachel’s project focuses on the specific interactions between Y. pestis and the protein product of this gene. One way of confirming the role of the protein in bacterial invasion is to see if the clustering of this protein around Y. pestis changes for cells overexpressing mutated versions of this gene. The extracellular portion of this protein contains several immunoglobulin (Ig)-like domains. Based on the preliminary data from an experiment comparing all proteins in the same family as the protein of interest, Rachel suspects that Ig-like domains 1, 2, and 3 affect bacterial attachment.
My project for the summer is to help design and build mutants that exclude each of the three domains. Infection of cells expressing the mutants can then show whether and how these Ig-like domains affect bacterial attachment and invasion. To accomplish this goal, I’ll first remove the DNA sequence coding for Ig-like domains 1, 2, and 3 from a plasmid that codes for the protein of interest. I’ll then ligate, separately, three mutated DNA fragments into the plasmid. HeLa cells transfected with the new plasmids will then express the mutated versions. Finally, infecting these HeLa cells with Y.pestis will reveal the critical domains for bacterial ligand binding. This project will contribute to the overarching goal of understanding how Y. pestis utilizes this protein to accomplish cellular invasion and how different genotypes of the protein affect Y. pestis infection.
