Using a Bacterial 2-Hybrid Assay to Investigate Bacteriophage Gene Function
Session Title
Science and Technology
College
College of Arts and Sciences
Department
Biology
Abstract
With up to 60% of bacteriophage genes having no predicted function, investigating gene characteristics could give novel insight into the role of these phage genes. In partnership with HHMI, Winthrop students have used a cytotoxicity assay to begin to characterize K cluster bacteriophage Cain’s genes. The inhibition of host growth indicates that the phage protein is likely interacting with a host protein to affect the host cell. The bacterial 2 hybrid (B2H) assay builds on these results by investigating which host protein(s) is targeted by the tested phage gene product. As a newly introduced assay to the program, Winthrop was charged with piloting the consistency of this procedure in university labs. Cain gp55, was the first gene to be cloned into the B2H expression plasmid. With the help of inducible promoters and reporter genes, evidence has shown that the Cain gp55’s expressed product interacts with mycobacterial Nus A; a protein known to be an important cellular transcription regulator. In our efforts to substantiate the B2H assay as consistent, we have tested a number of additional phage genes including Waterfoul gp47, Cain gp2, and Giles gp3. Since the most recent test with Giles gp3 yielded similar results to those at HHMI, we believe our B2H assay to be robust and ready to test more of Cain’s genes. Continuing to investigate these protein-protein interactions will give us insight into how Cain infects its host, replicates, and possibly protects its host from further phage attack or infection.
Start Date
15-4-2022 12:00 PM
Using a Bacterial 2-Hybrid Assay to Investigate Bacteriophage Gene Function
With up to 60% of bacteriophage genes having no predicted function, investigating gene characteristics could give novel insight into the role of these phage genes. In partnership with HHMI, Winthrop students have used a cytotoxicity assay to begin to characterize K cluster bacteriophage Cain’s genes. The inhibition of host growth indicates that the phage protein is likely interacting with a host protein to affect the host cell. The bacterial 2 hybrid (B2H) assay builds on these results by investigating which host protein(s) is targeted by the tested phage gene product. As a newly introduced assay to the program, Winthrop was charged with piloting the consistency of this procedure in university labs. Cain gp55, was the first gene to be cloned into the B2H expression plasmid. With the help of inducible promoters and reporter genes, evidence has shown that the Cain gp55’s expressed product interacts with mycobacterial Nus A; a protein known to be an important cellular transcription regulator. In our efforts to substantiate the B2H assay as consistent, we have tested a number of additional phage genes including Waterfoul gp47, Cain gp2, and Giles gp3. Since the most recent test with Giles gp3 yielded similar results to those at HHMI, we believe our B2H assay to be robust and ready to test more of Cain’s genes. Continuing to investigate these protein-protein interactions will give us insight into how Cain infects its host, replicates, and possibly protects its host from further phage attack or infection.
