Characterization of Growth for the Subcluster B5 Mycobacteriophage Donny Using Optical Density Measurements

Abstract

The mycobacteriophage Donny was discovered in Dahlonega, GA in 2016 as part of the SEA-PHAGES Program and is currently only one of seven verified phages in subcluster B5. Through various protocols for phage DNA purification and amplification, gene expression can be observed during lysis of its host, Mycobacterium smegmatis using real time PCR. Bacteriophages provide inherently novel subjects for genetic analyses as they are a highly diverse and ubiquitous group. There is little published research on phage from Cluster B which are a large yet genetically distinct group compared to other clusters or singletons. In order to begin analyzing gene expression during the phage life cycle, observation of Donny’s unique burst time is required so that optimal time frames can be selected for RNA extraction. Using absorbance data obtained from Optical Density (OD) measurements at a wavelength of 600nm, a potential time frame for RNA extraction during early, middle, and late infection was determined. This project contributes to the overall goal of characterizing the genes expressed during Donny's lytic cycle using primers designed from sequence analysis of all 96 genes.