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.
Methods
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.
• Both samples were filtered and 10µL of each sample
were added to Agar plates. In addition1mlof the host
cells Macrobacterium foliorum were added.
• Plaques form each sample were collected for each
sample. Due to the plaque morphology, the phage
from the Dog Park enrichment culture was selected.
• 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.
• The 10-4 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
C HotDog's plaques are circular in shape as a result of the
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
Results
For the HotDog bacteriophage the plaque morphology is
consistent with the typical standard form.
.
• 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.
Discussion
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.
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
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 Alliance-
Phage Hunters Advancing Genomics and Evolutionary Science