Date of Award
Master of Science
Steve Ripp, Todd Reynolds, David Golden
Detection of pathogenic bacteria in the environment and food products is of increasing importance especially in light of recent outbreaks of Escherichia coli O157:H7. I describe here a bacteriophage based bioluminescent bioreporter method for E. coli O157:H7 detection that combines the specificity bacteriophage have for their host, quorum sensing, and lux based bioluminescence from Vibrio fischeri. This new method for detection of E. coli utilizes the luxI/luxR quorum sensing present in V. fischeri which uses N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) as an autoinducer (Miller & Bassler 2001). Once the concentration of OHHL is high enough it binds the LuxR protein initiating transcription of luxCDABE and additional luxI, leading to the production of light. This bioluminescent bacteriophage bioreporter method uses 2 components, first, the bioreporter cell called E. coli OHHLux which carries the complete lux cassette (luxCDABE) along with the transcriptional regulator luxR (Ripp et al. 2006). The OHHLux (lambda phage resistant) detects the diffusible OHHL produced by the second component, the luxI engineered phage which when infecting target E. coli cells produces autoinducer.
The initial feasibility of the phage based reporter assay was tested using the well characterized lambda phage and E. coli K12 model (Ripp et al. 2006). Once the initial feasibility of this bacteriophage based bioluminescent bioreporter system was confirmed, an E. coli O157:H7 specific phage, PP01, was constructed with a luxI insert. This PP01- luxI phage was used in conjunction with E. coli OHHLux to test for E. coli O157:H7 in pure culture, apple juice, ground beef, tap water, and spinach rinsates. This method has the potential to be a sensitive, rapid, and non-invasive method of screening for E. coli O157:H7 contamination. The system provided rapid and sensitive detection with results in well under 24 h even at E. coli O157:H7 concentrations as low as 1 CFU/ml (Brigati et al. 2007, Ripp Submitted 2007). Our system is self-sufficient and could be adapted to be fully-automated and capable of high throughput screening in a production line setting.
Johnson, Courtney M., "Development of a Bacteriophage Based Bioluminescent Bioreporter System for the Detection of Escherichia coli K12 and O157:H7. " Master's Thesis, University of Tennessee, 2008.