Bacteria developed resistance to penicillin a mere four years after the groundbreaking antibiotic was first mass produced (Casadevall, 2010). Since then, the development of antibiotic-resistant bacteria has steadily risen, causing millions of difficult to treat infections annually. The challenge is to identify ways to combat these menacing microbes. Bacteriophages are viruses that infect bacteria and can potentially be used to eliminate deadly antibiotic-resistant bacteria. The number of sequenced bacteriophage genomes has increased tremendously over the past 10 years, but little is known about the function of most bacteriophage genes. The purpose of this study was to expand our understanding of bacteriophage gene function by identifying genes that are essential for lytic growth of bacteriophage HK639. Using recombineering, a genome engineering technique, five HK639 genes and one regulatory sequence were individually replaced with a kanamycin gene. The recombinants were selected on kanamycin plates, and the gene replacements were confirmed by Polymerase Chain Reaction (PCR). To determine if the replaced genes and regulatory sequence were essential for lytic growth, the recombinants were tested for the release of viable phage. Phage production was unaffected by four of the gene replacements, which indicates the replaced genes are not essential for lytic growth. However, replacement of ORF 40 prevented phage growth. These results indicate that ORF 40 performs an essential function. Biochemical analysis of these proteins will be necessary since sequence analysis did not reveal any clues about the potential function of ORF 40.
Advisor(s) or Committee Chair
Rodney King, Ph.D.
Bacteriology | Biology | Microbiology | Virology
Carroll, Amber, "Identifying Essential Viral Genes through Genomic Engineering" (2020). Mahurin Honors College Capstone Experience/Thesis Projects. Paper 892.