Creation of a Recombinant Bacteriophage to Express Beta-Galactosidase http://life.anu.edu.au/viruses/ICTVdB/em_t4.gif Catherine Swedberg & Heather Talbott with Dr. John Willford Department of Microbiology and Molecular biology University of Wyoming

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

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

Bacteriophage Viruses that infect bacteria Very host specific Bind to receptors on host cell membrane Can be used for detection of bacteria http://www.nsf.gov/od/lpa/news/02/pr0207images.htm http://en.citizendium.org/wiki/Bacteriophage

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

Lytic Cycle in Wild Type

Lytic Cycle after recombination

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)

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

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 http://www.sigmaaldrich.com/thumb/prodimages/b/b3928.jpg

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

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

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

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

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

Future Research Successfully introduce reporter gene into T4K10 bacteriophage Re-run Phast Swab Assay with new reporter phage http://engineering.curiouscatblog.net/images/bacteriophage.jpg

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

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