Isolation and Analysis of a Mycobacteriophage Specific to Mycobacterium Smegmatis

Abstract

BACKGROUND: Bacteriophages are viruses that kill bacteria. It is estimated that there are roughly 1031 types of bacteriophages in the world, and that every bacterium has an average of 10 bacteriophages that can infect it. A mycobacteriophage is a type of bacteriophage which has a mycobacterial species as its host. There are 30 known clusters, or types, or mycobacteriophages which all contain very distinct genetic structures, but there are still countless mycobacteriophage populations that need to be isolated. Now that microorganisms are becoming resistant to many antibiotics, the study of bacteriophages is increasingly important because they have an incredibly untapped potential to treat resistant bacteria, eradicate bacterial contaminants in food products, promote ulcer healing, control bacterial growth during fermentation, and much more. The goal of this project was to discover new bacteriophages that might contribute to novel medical solutions. MATERIALS AND METHODS: We plated 29 agar plates for 8 different samples. One sample resulted in plaque growth, from which we performed 9 streak tests, underwent a dilution series and filtration to eliminate the contamination, and harvested a High Titer Lysate. After isolating and purifying the phage genomic DNA, we analyzed the sample and quantified the DNA using a Nanodrop spectrophotometer, and we used agarose gel electrophoresis to separate and analyze the DNA fragments. Finally, we used electron mycrocopy to visualize the physical structure and confirm the successful isolation of a single mycobacteriophage population. DISCUSSION: The cloudiness of the plaque produced by the bacteriophage suggests it is a temperate phage, meaning it switchs between two behavior types regarding replication and survival. Analysis of the physical structure of the mycobacteriophage reveals more information about its genomic DNA; its large capsid diameter suggests lengthy indwelling genomic DNAand its relatively long tail suggests a large tape-measure gene. We were unble to analyze the agarose gel electrophoresis results due to time constraints. However, the results of the genomic DNA sequence will hopefully prove that our bacteriophage is a distinct population. CONCLUSION: We successfully captured, isolated, and purified a single bacteriophage species from the local environment of West Lafayette, Indiana. Upon isolating a single bacteriophage species, we then isolated, purified, restricted, and analyzed the genomic DNA of the phage. Finally, we documented our findings on the Bacteriophage Database as FelixElFago (http://phagesdb.org/phages/FelixElFago/) and sent our DNA to the Sequencing Center.