phage poster

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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

.