Abstract

Introduction

Experimental Design

Results Conclusions

Characteristics of Gordonia terrae Mycobacteriophages Fetty and WaapKarthik 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. "DNASequence, 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 SampleMake a

Direct PlateMake an

EnrichmentPerform 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.

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