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RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
Using Bacillus thuringiensis as a host, bacteriophage was isolated
and characterized from the surface of a sandbox located in the
Merion in Columbia, Maryland. Ophiuchus shows a clear plaque
morphology which lead us to the assumption that we had a phage
with a lytic cycle. By measuring the tail and head length, as well as
finding a contractile tail in our electron micrographs and therefore
we were able to identify our phage as being part of the Myoviridae
family. Further assays showed that our phage has a genome size of
39.3 KBp. The PCR analysis showed that our phage had primer
hits that belong in clusters 2B and 3A.
ABSTRACT
INTRODUCTION
METHODS AND RESULTS
Table 2.
The restriction gel was used for this data present in table 1,
and it showed that there were multiple enzyme cuts in lanes
4,5 and 7. Lane 4, which was where Cla I restriction
enzyme is located has the highest bands counted.
OPHIUCHUS VS. 59 PHAGESTable 3. Host Range Data – shows the types of strains
our phage can infect.
REFERENCES
Trauner, Andrej, Sonia Borrell, Klaus Reither, and Sebastien Gagneux. "Evolution of Drug
Resistance in Tuberculosis: Recent Progress and Implications for Diagnosis and Therapy." National
Center for Biotechnology Information. U.S. National Library of Medicine, 25 June 2014. Web. 20 Nov.
2014. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4078235/>.
Worthington, Roberta J., and Christian Melander. "Combination Approaches to Combat
Multi-Drug Resistant Bacteria." National Center for Biotechnology Information. U.S. National Library
of Medicine,. N.p., 18 Jan. 2013. Web. 20 Nov. 2014.
<http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC3594660%2F
SEA – Phage Hunters , BIOL302 Lab Genetics Manual
ACKNOWLEDGEMENTS
• Ralph Murphy – preparing all the materials we needed for our experiments
• Chere Petty – helping us obtained TEMs and teach us how to use an EM
• Dr. Steven Caruso – Giving us the opportunity to isolate and characterize our own phage
• Ling Cao - Our awesome section Teaching Assistant
• William Larson Angel – Undergraduate TA
In the health field, the use of antibiotics as a treatment for bacterial
and viral infections has seen a sharp increase in the demand for the
discovery of new antibiotics. Microphages and bacteriophages are
the driving force behind antibiotics responsible for the treatment of
viral or bacterial infections, respectively. It is of great importance
to know not only which microphage or bacteriophage can be used
for which infections but also if the treatment will have any effect.
Recently there has been an increase in multi-drug resistant
organisms, more commonly bacteria. Mycobacterium tuberculosis
exemplifies such a multi-drug resistant bacterium that has evolved
because of resistance-conferring mutations on its chromosome.1 In
another similar story, the more recent introduction of daptomycin
in 2003, has allowed the bacterium Enterococcus faecium to find
resistance to the antibiotic.2 It is for these reasons why it is
important to find and isolate novel phages. The uniqueness of
novel phages ensures better treatment of bacterial infections since
the bacteria has never encountered the phage or had time to
overcome the effects of the phage through evolution.
BIOL 302L, Department of Biological Sciences
University of Maryland, Baltimore County , Baltimore , MD
Russell Ampofo & Susana S.Hoyos
Phages on the Playground: The Role of Bacillus thuringiensis in Isolating a Novel Phage
A B
E
DC
Figure 3. Plaque Morphology
A titter assay was done after successfully isolating a phage using
the 3x streak technique. The results displayed that the titer was
5.8 x 10E 7 pfu/mL and that our plaques were clear, round with
fuzzy edges around the clear center.
Figure 4. TEMs (A-E): Using Electron Microscopy , electron micropgrahs
were made and quantified. From the pictures we were able to specifically
believe that Ophiuchus belonged to the Myoviridae family. A phage from the
Myoviridae shows characteristics unique to its own category, such as a
contractile tail, which is shown in Figure 2D.
Legend:
1: B. cereus, 4: B. substillis, 5: B. Cereus Gibson 971, 6: B. thuringiensis p552A1, 7: B. thuringiensis
subsp. Berliner 1915, 8: B. thuringiensis subsp. Kurstaki, 9: B. simplex RWR-2, 10: B. megaterium,
JJV118, 11: B. cereus 2013 , 12: B. subtillis subs. Spizizenii, 13: B. cirulans Jordan, 14: B. subtillis SMY,
15: B. linchenformis A5, 16: B. thuringiensis subsp. Al Hakam ,17: B. cereus 14579,18: B. anthracis
deltesterne, 19: B.pumillus 113, 20: B. megaterium ,21: B. cereus FDA5, 22: B. globegil ,23: B.
weinstephanesis
Figure 3a. Legend:
1 = standard ladder
2 = undigested DNA
3 = Bam HI
4 = Cla I
5 = Eco. RI
6 = Hae III
7 = Hind III
8 = Sma I
= Multiple Enzyme cuts
Figure 5. Digestion Gel
To analyze the genome of Ophiuchus DNA, gel electrophoresis
was done using restriction enzymes. What we found that our DNA
was not cut by Bam HI, Hae III and Sma I enzymes.
Table 1. This table displays the measurements taken from each
electron micrograph. Phage D is the phage with a contractile tail,
and is significantly different from the rest of the measurements.
Figure 6. PCR Gel
A PCR generates thousands of copies from a single template of
DNA. This test was used to identify and get an idea of what other
phages it may relate to by using primers from particular locations.
The results for Ophiuchus DNA had similar sequences that
belonged in clusters 2B and 3A.
Legend:
Green = Cluster 1 (C1)
Pink = Cluster 2 (C2)
Yellow = Cluster 3 (C3)
= C1-A
= C1-B
= C2-A
= C2-B
= C3-A
= C3-B
Graph 1.
Standard curve
made from our
digestion gel was
use to estimate
Ophiuchus genome
size.
Genome size= 39.3
KBp
Figure 2. Lytic vs. Temperate
Phage life cycle
Figure 1. Phage Structure
Both Figure 1 and 2 were taken from BIOL 302
Lab Genetics Manual
Graph 2. Phage Measurements compared to the rest of the class
.
Graph 3. Morphotype compared to
the rest of the class
.
Table 4. Restriction Enzyme Cuts compared to the rest of the
class
.
Graph 2. Phage Measurements compared to the rest of the class
.
Table 5. Primer Hits
compared to the rest of the class
.
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