Bacteriophage Discovery

Discovery of HotDog Phage Emilie Colon and LaNeigh Jones Department of Biology, Radford University, Virginia

Introduction Bacteriophages are viruses that specifically infect bacterial cells. Theses are thought to be widely distributed since their niche is found everywhere bacteria are located. The lack of knowledge and research in the field initiated the SEA-PHAGES program through the Howard Hughes Medical Institute. The purpose of this project is to discover phages that have not yet been already discovered. We practiced the use of direct isolation and enrichment cultures to help us discover our phage morphology. The purpose of seeing the morphology allows us to see the unique differences in plaque and make dilutions for the steps of purification of the phage so it can be sequenced.

Results 10-4

• The plate was flooded with phage buffer and filtered after 2 hours. From the solution obtained a new 10-8 serial dilution was conducted . • Number of viable plaques were counted and a titer was identified. The plate with the best titer was flooded, to further amplify the phage. After repeated steps of dilutions, enrichments, and our final titers, we then did our TEM, our DNA Phage extraction and Restriction Enzyme Digest to begin analyzing the DNA.

Figures A

B

For the HotDog bacteriophage the plaque morphology is consistent with the typical standard form. .

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The plaques were commonly circular in shape and unclear. The plaques were also well defined in shape. These plaques take a longer period to form, after a day there were no visible plaques. The plaques typically were visible after two to three days.

• The spot titer test indicates an approximate value of phage concentration per volume. • The images receive from the Transmission electron microscopy (TEM) showed a clear view of HotDog phage. The phage has an icosahedral capsid, a long neck, and short tail fibers.

Methods

Discussion

We first began having to collect samples, which we received from the Dog Park as well as Radford’s Wetlands. •

To Isolate bacteriophage a direct isolation and enrichment culture were conducted.

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Both samples were filtered and 10µL of each sample were added to Agar plates. In addition1mlof the host cells Macrobacterium foliorum were added.

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Plaques form each sample were collected for each sample. Due to the plaque morphology, the phage from the Dog Park enrichment culture was selected.

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Once selected a spot test was conducted, with a 10ˉ⁸ concentration set up for serial dilutions.

• Based on observations of the dilutions, the concentration was lowered 10ˉ⁶. We picked a plaque from the 10-3 concentration and did dilutions up to 10ˉ⁶ concentration.

C

Based upon our successful project, we were able to successfully isolate a phage from the environment. Hotdog bacteriophage has a slow lytic cycle because plaques developed after the standard period. HotDog's plaques are circular in shape as a result of the infection pattern. Once a single phage infects M. foliorum host cells and the other phages are released; they all diffuse and infect cells in close proximity. The overall structure of HotDog looks similar to what we expected based on general knowledge of phage morphology. The short tail fibers indicate that the phage belongs to Caudovirale family.

References Figure A shows the plaques formed from the enrichment culture. The sample was collected at the local dog park (latitude: 37.143° and longitude: -80.546). Results obtained from the spot titer are shown in figure B. Figure C shows the TEM image of the isolated phage

Nicholas M. I. Taylor, Nikolai S. Prokhorov, Ricardo C. Guerrero-Ferreira, Mikhail M. Shneider, Christopher Browning, Kenneth N. Goldie, Henning Stahlberg, Petr G. Leiman. Structure of the T4 baseplate and its function in triggering sheath contraction. Nature, 2016; 533 (7603): 346 SEA PHAGES Manual. Howard Hughes Medical Institute. The Science Education AlliancePhage Hunters Advancing Genomics and Evolutionary Science