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  • Abstract

    Introduction

    Experimental Design

    Results Conclusions

    Characteristics of Gordonia terrae Mycobacteriophages Fetty and WaapKarthik Chinnappan and Gina DIncau, 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. Waaps DNA was digested with five restriction

    enzymes and run on a 0.7% agarose gel. HaeIII most noticeably cut Waaps DNA into many small

    fragments. The other four enzymes did not make significant cuts in the

    DNA.

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