Download Creation of a Recombinant Bacteriophage to Express Beta

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

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

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)

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
 Stable in cell lysates
 Previously used to construct
successful reporter phage

Easy to measure
 3 substrates may be used
 Colormetric
 Luminescent
 Fluorescent
http://www.sigmaaldrich.com/thumb/prodimages/b/b3928.jpg

Colormetric substrate, X-gal,
was used
Reporter Phage Construction

Bacteriophage T4K10 is already denAand denB Both are non-essential genes
 Selected denA as insertion site

Gene 22 promoter
 Late, strong promoter
 Lots of protein production

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

Dr. Gerry Andrews

MOLB Department
 Karen White

INBRE Undergraduate Research Program

Dr. John Willford
Thank
you!
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Questions….
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