<p>The long-term goal is to sensitively detect very low concentrations of pathogenic foodborne bacteria that negatively impact public health. </p><p>The short-term goal is utilize genetic engineering techniques to increase expression of alkaline phosphatase to facilitate single organism detection. </p><p>Develop a molecular biological strategy using CRISPR/Casto genetically modify bacteriophage T7.2. Develop a strategy using homologous recombination to genetically modify bacteriophage T7.3. Develop a strategy using restriction enzyme digests to genetically modify bacteriophage T7.4. Utilize CRISPR/Cas and/or colorimetric substrate technology for selection of desired recombinants. 5. Optimize reporter gene expression by varying insert placement in T7 phage genome.6. Optimize reporter gene expression by introducing genetic insert concatamers to produce multiple copies at once.7. Purify and characterize reporter enzymes.8. Remove phage replication abilities. </p>
Foodborne pathogens create enormous societal costs as a result of their ability to cause significant human suffering and loss of life. The top 14 foodborne pathogens in the United States create almost 9 million infections per year. Detection of these harmful microorganisms is paramount to our strength as a society. Our long-term goal is the improvement of food safety and animal health through improved pathogen detection systems. Our approach for improving pathogen detection systems will involve utilizing newly developed molecular biological techniques to genetically modify a virus that only infects bacteria (bacteriophage). This genetic modification will force target pathogens (upon phage infection) to express a unique reporter enzyme as a positive indicator for the presence of pathogens. Optimizing reporter enzyme expression levels will allow for lower bacterial detection limits to be reached. Phages incapable of replication will be constructed to preserve the integrity of the cell and to minimize the potential of environmental contamination. A major goal of this project is to reproducibly detect pathogenic foodborne bacteria in concentrations less than the required dose for illness. The ultimate goal is to design a detection scheme capable of single organism detection that will provide compliance with many zero-tolerance regulations. This project will dramatically improve the capabilities of low cost diagnostic platforms.