1. Creation of a Recombinant Bacteriophage to Express Beta-Galactosidase
Catherine Swedberg & Heather Talbott with Dr. John Willford
Department of Microbiology and Molecular biology
University of Wyoming

2. Food-borne illness
Pathogens such as Escherichia coli, Salmonella, Listeria, and Camplyobacter are a major cause of food-borne illness
Estimated that there are 9.4 million cases of food-borne illness in the USA annually
55,961 hospitalizations annually
1,351 deaths annually
(Center for Disease Control, 2011)
Huge impact on public safety and economic impact on food industry

3. Current Methods of Detection
Current methods detecting pathogens in food use several techniques
Classic microbiological methods
Serological methods (ELISA)
Genetic methods (PCR)
Potential issues
Long detection time
Labor intensive process
Lack of sensitivity

4. Bacteriophage Viruses that infect bacteria Very host specific
Bind to receptors on host cell membrane
Can be used for detection of bacteria

5. Reporter Phage
Reporter phage have successfully detected bacteria such as Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7 and Salmonella spp.
Reporter Genes used:
Luciferase (lux) genes
Ice nucleation (inaW) genes
Beta-galactosidase (lacZ) genes

6. Lytic Cycle in Wild Type

7. Lytic Cycle after recombination

8. Previous Reporter Phage Study
Reporter phage with lacZ inserted into uvsY gene
uvsY gene is responsible for DNA replication, repair, and recombination
An essential gene
Found reporter phage had longer replication times and severely reduced burst size
Wild type 132 virus burst size
Reporter phage 4 virus burst size
Utilized in the Phast Swab Detection System with moderate success (Willford & Goodridge, 2008)

9. Project Objections Fix problems associated with the previous study
Choose a non-essential gene to insert the reporter gene
Create plasmid based on new reporter insertion design
Produce new reporter phage

10. Beta-galactosidase Beta-galactosidase Easy to measure
Stable in cell lysates
Previously used to construct
successful reporter phage
Easy to measure
3 substrates may be used
Colormetric
Luminescent
Fluorescent
Colormetric substrate, X-gal,
was used

11. Reporter Phage Construction
Bacteriophage T4K10 is already denA- and denB-
Both are non-essential genes
Selected denA as insertion site
Gene 22 promoter
Late, strong promoter
Lots of protein production

12. Created in Previous Work
pDen6
Plasmid containing the denA gene
pEF25
Plasmid containing the P22::lacZ gene fusion
Need to excise the fusion and insert it into the denA gene

13. Plasmid Creation pDen6 cut with SnaBI pEF25-75 cut with BsrBI and SmaI
800 bp with P22::lacZ fusion ligated into cut pDen6

14. Plasmid Results Ligation was transformed into E. coli HB101
Plasmid preps run on a gel
up-shift from pDen6 present

15. Reporter Gene Confirmation
Multiplex PCR with DenAF, DenAR, p22F, and lacZR primers
Awaiting sequencing confirmation

16. Future Research
Successfully introduce reporter gene into T4K10 bacteriophage
Re-run Phast Swab Assay with new reporter phage

17. Acknowledgements Dr. Gerry Andrews MOLB Department
Karen White
INBRE Undergraduate Research Program
Dr. John Willford

18. Thank
you! ? ? ? ? ? ?
Questions…. ? ? ?