1. Confirmation of the Gene Cassette Swap of AR1 Tail Fibers to T4K10 Bacteriophage Using Genotypic and Phenotypic Methods Kassandra Willingham Department of Microbiology April 30, 2010 Mentor: Dr. John Willford

2. An Introduction to Bacteriophage  Viruses that specifically infect bacteria  One of the most prevalent biological entities  Key term- host range: the specific bacteria that a bacteriophage is capable of infecting  Typically very specific

3. The Importance of Host Range  Host range is determined by the ability of the phage’s tail fiber to bind with its host’s surface receptors.  i.e., modification of the phage’s tail fiber sequence will subsequently modify the phage’s host range.  A previous study involved isolation of the tail fiber gene cassette of bacteriophage AR1 and recombining it into bacteriophages T4K10 and T2.  Yet to be fully confirmed.

4. Swapping Tail Fibers Bacteriophage picture adapted from: The Internet Encyclopedia of Science

5. Swapping Tail Fibers Bacteriophage picture adapted from: The Internet Encyclopedia of Science

6. Research Objectives  Characterize the host range of the potential recombinant phages (ARX) as compared to the initial phages (T4, T2, & AR1).  Conduct Restriction Fragment Length Polymorphisms (RFLPs) on phage DNA to characterize genotypically.

7. Host Range Typing Methods  Phage were amplified and enumerated  All were >10 7 pfu/mL  10 µL of phage lysate was dropped on to the surface of solidified top agar containing 200 µL of each bacterium  151 strains of Escherichia coli, 10 negative control species  The ability of phage to lyse the host is then recorded using “complete” [lysis], “partial,” and “none.”

8. T4K10T4ARX1 T4ARX2T4ARX3 T2T2ARX1 T2ARX3AR1 T2 and T4K10 exhibit complete lysis Recombinant phages T4ARX1, 2, 3, and T2ARX1, and 2 exhibit partial lysis AR1 does not exhibit lysis. (No visible clearing zone.) 10 µL phage lysateLawn of bacterial growth

9. Host Range Typing Results PhageGood or Partial Clearance T4K1032 T4 ARX1101 T4 ARX299 T4 ARX3103 T2102 T2 ARX194 T2 ARX297 AR183  43 of the 46 E. coli strains infected by AR1 but not T4K10 were infected by the T4 ARX phages (at least partially)  T2 has a desirable natural host range

10. Species /strain T4K10T4ARX1T4ARX 2 T4ARX 3 T2T2ARX 1 T2ARX 2 AR1 EC 38 nonecomplete partialcomplete w/some resistance complete 14 nonecomplete partialcomplete 24A nonecomplete nonecomplete 828 nonepartial partial- almost complete complete 5 nonecomplete nonecomplete LG37 nonecomplete nonecomplete Table 1. Escherichia coli Strains Exhibiting a Successful Exchange

11. Use of Controls  Positive control: E. coli CR63  Previous experiments show that T4 and T2 phage are capable of lysing E. coli CR63, but AR1 is not.  Host range data showed that this control was successful, i.e. ARX phages can still partially infect E. coli CR63  Negative controls: Gram-positive bacterium Staphylococcus aureus, non E. coli Gram-negatives  T4K10, T2, and AR1 are generally unable to infect non-E. coli Gram-negatives or Gram-positives.  Host range data from this experiment showed that all negative controls except for Shigella dysenteriae were resistant to infection by T4, AR1, and the recombinant phages.

12. Results: Restriction Fragment Length Polymorphisms  In silico experiments worked great.  Provided potential successful enzymes to utilize  HpaI, XbaI, SmaI  T4K10, AR1, and the recombinant T4ARX strains were not successfully cut by the RFLPs used.  2, 4, & 24 hour incubations  All enzymes identified in silico cannot cut methylated DNA  Currently found in the Box of Shame

13. Discussion  Host range results support the supposition that the tail fibers from AR1 were successfully swapped into T4K10 and T2.  Genotypic characterization was unsuccessful.  Along with previous physical characterization, we are closer to confirming tail fiber modification.

14. Future Experiments  Re-running host range studies on results that were problematic.  A small percentage of data showed extreme resistance in E. coli strains that should have exhibited at least partial sensitivity.  Finding an RLFP that can cut T4K10 and recombinant phages DNA.  PCR is a more likely candidate for genotyping.

15. References  Bacteriophage. New Horizons Diagnostic Corporation. N.p., n.d. Web. 11 Jan. 2011..  Bacteriophage. Meyer, Gene. Microbiology and Immunology Online. University of South Carolina School of Medicine, n.d. Web. 11 Jan. 2011..  Development of a field-based assay for rapid detection of enterohemorrhagic Escherichia coli (EHEC). Willford, J. Ph.D. dissertation, University of Wyoming, United States -- Wyoming. Dissertations & Theses @ University of Wyoming. (Publication No. AAT 3338821). 2008.

16. Acknowledgements  Dr. Gerry Andrews  Molecular Biology Department  Karen White  INBRE Undergraduate Research Program

17. Questions ?