Study and Utilization of the K2 Mycobacteriophage Findley as an Antibacterial Agent

Abstract

This project focuses on bacteriophages, viruses that infect host bacterial cells. When infected, the host bacterial cell will either enter the lytic or lysogenic phase. In the lytic cycle, the host cell will replicate phage DNA and create new phages. The cell will lyse to release the newly created phages. During lysogeny, the phage genome is integrated into the bacterial cell genome. New phages are not produced, and the host cell does not lyse. Specifically, this project focuses on mycobacteriophages, phages which infect bacteria of the genus Mycobacterium. In particular, we are looking for phages that will infect the host bacterium Mycobacterium tuberculosis, so that they might serve as a clinical therapy to fight tuberculosis infection. To do so, our goal is to genetically modify K2 phages, which are known to infect M. tuberculosis. I have focused on the K2 phage Findley, isolated by the SEA PHAGES program at Nyack College in Nyack, New York. Initially, we purified a Findley lysogen, which is a bacterial host cell with the Findley phage DNA incorporated into the bacterial cell's chromosomal DNA. In order for a phage to enter the lysogenic cycle, and for its DNA to be incorporated into a host genome, the phage must contain an integrase gene. The integrase gene determines where the phage DNA will be incorporated into the host genome, through a method called site‐specific recombination. We confirmed the existence of a Findley lysogen through a method of Polymerase Chain Reaction (PCR) which exploits site‐specific recombination. I used a series of primers to amplify certain segments of DNA at the exact points of recombination in the lysogen genome. The presence of these certain amplicons confirmed that lysogeny did occur, and that the integrase gene is functional in the Findley phage.After confirming the functionality of an integrase gene, the next goal of our project was genetic modification of the Findley phage. The goal of genetic modification is to change the phage so that it more efficiently infects and lyses the host Mycobacterium cells. To do so, we aim to knock out one of the genes responsible for the lysogen phase, the repressor gene. The repressor gene dictates that the bacterial cell be immune to further infection from the same phage and the cell becomes a lysogen. During lysogeny, the cell is not lysed. However, if a phage lacks this gene, it will only enter the lytic phase, making it a “killing” phage only. To construct this mutant phage, we performed a method of allelic exchange to delete the gene, called the BRED (Bacteriophage Recombineering with Electroporated DNA) method. The allelic exchange results in a specific portion of the phage genome being deleted. We designed a substrate DNA that is identical to regions of the phage genome, but with the specified gene missing. We used electrocompetent Mycobacterium smegmatis cells to perform the transformation. During the transformation, wild‐type phage DNA, the substrate DNA and electrocompetent M. smegmatis cells were electroporated so that the host bacterial cell will take both types of the DNA and recombine it. The M. smegmatis cell then produced the mutant phage during a lytic cycle. Plating the electroporated cells with host cells results in plaques that can be screened for phage with the desired deletion.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.