Transcript Confirmation of the Gene Cassette Swap of AR1 Tail Fibers to

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
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
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.
Swapping Tail Fibers
Bacteriophage picture
adapted from: The
Internet Encyclopedia of
Science
Swapping Tail Fibers
Bacteriophage picture
adapted from: The
Internet Encyclopedia of
Science
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.
Host Range Typing Methods
 Phage were amplified and enumerated
 All were >107 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.”
10 µL phage lysate
Lawn of bacterial
growth
T4K10
T4ARX1
T2
T2ARX1
T4ARX2
T4ARX3
T2ARX3
AR1
• 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.)
Host Range Typing Results
Phage
Good or Partial Clearance
T4K10
32
T4 ARX1
101
T4 ARX2
99
T4 ARX3
103
T2
102
T2 ARX1
94
T2 ARX2
97
AR1
83
 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
Table 1. Escherichia coli Strains
Exhibiting a Successful Exchange
Species
/strain
T4K10
EC 38
none
14
T4ARX1
T4ARX
2
T4ARX
3
T2
T2ARX
1
T2ARX
2
AR1
complete
complete
complete
partial
complete
w/some
resistance
complete
complete
none
complete
complete
complete
partial
complete
complete
complete
24A
none
complete
complete
complete
none
complete
complete
complete
828
none
partial
partial
partial
partialalmost
complete
complete
complete
complete
5
none
complete
complete
complete
none
complete
complete
complete
LG37
none
complete
complete
complete
none
complete
complete
complete
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.
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
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.
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.
References
 Bacteriophage. New Horizons Diagnostic Corporation.
N.p., n.d. Web. 11 Jan. 2011.
<http://www.nhdiag.com/phage.shtml>.
 Bacteriophage. Meyer, Gene. Microbiology and
Immunology Online. University of South Carolina School of
Medicine, n.d. Web. 11 Jan. 2011.
<http://pathmicro.med.sc.edu/mayer/phage.htm>.
 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.
Acknowledgements
 Dr. Gerry Andrews
 Molecular Biology Department
 Karen White
 INBRE Undergraduate Research Program
Questions?