1. Abstract
Introduction
Experimental Design
Results Conclusions
Characteristics of Gordonia terrae Mycobacteriophages Fetty and Waap
Karthik Chinnappan and Gina D’Incau, University of Pittsburgh Dept. of Biological Sciences
It is important to analyze mycobacteriophages because they have
the potential to combat drug resistant bacteria and provide other useful
applications in the field of bacteriophage genomics.
Mycobacteriophages Fetty and Waap originated from the same soil
sample collected in Pittsburgh, PA. Using a variety of methods, both
the phages Fetty and Waap were identified with the use of Gordonia
terrae 3612 host cells. Once morphologies were separated, the phages
were then plated so that only one morphology was present on each
plate for further analysis. The lysates were used for DNA extraction,
serial dilutions for tittering, and electron microscopy (EM) grids. The
DNA extraction from Waap then underwent restriction enzyme digest
with five different enzymes.
● Bacteriophages are viruses that infect bacteria that need a bacterial
host cell to replicate their DNA.
● Mycobacteriophages are viruses that infect mycobacterial host cells,
such as Gordonia terrae 3612, and have a tremendous genetic diversity
and abundant presence in the environment.
● We study bacteriophages because they are easy to work with and they
thus provide a gateway into scientific research.
● Phages carry a diverse and abundant population and therefore have
the potential to serve as therapeutic agents in bacteriophage therapy.
● We hope to isolate, examine, and characterize phages into specific
phage clusters, learn more about Gordonia terrae host cells by seeing
which phages infect and observe what this phage can teach us about
viruses and what role it plays in viral therapy.
● Our research supports the idea that
mycobacteriophages are genetically very
diverse have many different morphologies.
● Our research allows for the addition of more
information in regards to the Gordonia terrae
phages and their characteristics that can later
be manipulated and utilized for other uses.
● Due to a lack of sequenced Gordonia terrae
phages, mycobacteriophage Waap has not
been named to a cluster yet.
References
Hatfull, Graham, and Roger Hendrix.
Mycobacteriophage Database. Pittsburgh Bacteriophage
Institute. Web.
Hatfull, Graham F., and Gary J. Sarkis. "DNA
Sequence, Structure and Gene Expression of
Mycobacteriophage L5: A Phage System for Mycobacterial
Genetics." Molecular Microbiology (1993): 395-405. Print.
Pedulla et al. “Origins of Highly Mosaic Mycobacteriophage
Genomes.” Cell (2003): 171-182. Print.
“SEA-PHAGE Lab Manual” Phage Hunting Program, 2015.
Figure One:
Mycobacteriophages Fetty and Waap Plaques on Gordonia
terrae
A. B.
Figure Two:
Mycobacteriophages Fetty and Waap in an Electron Microscopy
A. B.
Figure Three:
Mycobacteriophage Waap in Agarose GelCollect a
Soil Sample
Make a
Direct Plate
Make an
Enrichment
Perform a
Spot Test
Make Serial
Dilutions
Quick and
Dirty Plate
of High
Titer Lysate
Concentrate
LysateDNA
Extraction
Restriction
Enzyme
Digest
Electron
Microscopy:
Negatively-
Stained Grid
Future Directions
● We hope to determine which cluster the
mycobacteriophages Fetty and Waap belong to.
● We aim to examine the genes of the phages
● Looking at these genes, we want to see if they can
play significant roles in bacterial infections and
human diseases.
Figure 1a. The plaques of figure
1a are round and medium sized
with cloudy halos surrounding a
clear center and have formed on
the Gordonia terrae bacterial
lawn.
Figure 1b. The plaques of figure
1b are small, turbid pinpricks
with a clear center that are
surrounded by a halo on the
Gordonia terrae lawn.
Figure 2a. The phage in
figure 2a has a long, flexible
tail that is 279nm and an
isometric head with positive
staining that is 64nm in
diameter.
Figure 2b. The phage in
figure 2b has a long, flexible
tail that is 261nm and a
round head with negative
staining that is 56nm in
diameter.
Figure 3. Waap’s DNA was
digested with five restriction
enzymes and run on a 0.7% agarose
gel. HaeIII most noticeably cut
Waap’s DNA into many small
fragments. The other four enzymes
did not make significant cuts in the
DNA.
Ladder
UncutDNA
BamHI
ClaI
EcoRI
HaeIII
HindIII