Isolation and Characterization of Waits, an A15 Subcluster Actinobacteriophage
Abstract
Isolation and Characterization of Waits, an A15 Subcluster Actinobacteriophage Audrey Waits and Indiren Pillay Bacteriophages are incredibly abundant viruses that exclusively infect bacteria. With estimated abundance at 1031, most bacteriophages have not been characterized. The goal of this study was to isolate and characterize a novel Gordonia terrae bacteriophage from Middle Georgia. Soil samples were collected from Lake Laurel and the Oconee River Greenway, enriched with Gordonia terrae and then filtered to test for phage activity. Prospective lysate was serially diluted and assessed for plaque formation, indicating phage activity. Plaques were then purified using serial dilutions until all plaques were the same diameter, suggesting a pure phage culture. Once purified, samples of phage were visualized by transmission electron microscopy. Phage DNA was extracted, purified and subjected to restriction enzyme digest using BamHI, ClaI, EcoRI, HaeIII, and HindIII and gel electrophoresis. The resultant DNA profile was then compared to other known digests of phage DNA. When no similar patterns were found, a concentrated DNA sample was then sent off for sequencing. After sequencing, DNAMaster and Phamerator software were used to annotate the genome. Electron microscopy revealed an icoshohedral head with average diameter for the capsid at 55nm and average length of the non-contractile tail at 125nm. Based on the physical characteristics, phage “Waits” is in the Siphoviridae family. Gel electrophoresis showed that BamHI, ClaI, and HaeIII cut the DNA. Genome sequencing resulted in 52856 bp with GC content 62%. Preliminary analysis indicates phage Waits is a novel bacteriophage in the A15 taxonomic subcluster with 101 genes.
Isolation and Characterization of Waits, an A15 Subcluster Actinobacteriophage
Isolation and Characterization of Waits, an A15 Subcluster Actinobacteriophage Audrey Waits and Indiren Pillay Bacteriophages are incredibly abundant viruses that exclusively infect bacteria. With estimated abundance at 1031, most bacteriophages have not been characterized. The goal of this study was to isolate and characterize a novel Gordonia terrae bacteriophage from Middle Georgia. Soil samples were collected from Lake Laurel and the Oconee River Greenway, enriched with Gordonia terrae and then filtered to test for phage activity. Prospective lysate was serially diluted and assessed for plaque formation, indicating phage activity. Plaques were then purified using serial dilutions until all plaques were the same diameter, suggesting a pure phage culture. Once purified, samples of phage were visualized by transmission electron microscopy. Phage DNA was extracted, purified and subjected to restriction enzyme digest using BamHI, ClaI, EcoRI, HaeIII, and HindIII and gel electrophoresis. The resultant DNA profile was then compared to other known digests of phage DNA. When no similar patterns were found, a concentrated DNA sample was then sent off for sequencing. After sequencing, DNAMaster and Phamerator software were used to annotate the genome. Electron microscopy revealed an icoshohedral head with average diameter for the capsid at 55nm and average length of the non-contractile tail at 125nm. Based on the physical characteristics, phage “Waits” is in the Siphoviridae family. Gel electrophoresis showed that BamHI, ClaI, and HaeIII cut the DNA. Genome sequencing resulted in 52856 bp with GC content 62%. Preliminary analysis indicates phage Waits is a novel bacteriophage in the A15 taxonomic subcluster with 101 genes.