The goal of this research is to harness the power of bacterial phage display to develop a biological amplifier for the detection of small numbers of pathogenic organisms in potable water and foods. The basic approach consists of the insertion of a second copy of a gene coding for a surface protein modified to express a predetermined binding epitope. Amplification of the recombinant phage is performed in a genetically modified host which suppresses expression of the modified surface protein. Subsequent infection of a wild type host using the modified parent phage results in progeny which are distinguishable from the parent and can be detected using immunoassays. Proof of principle experiments of this technology utilizes M13, an E. coli F' bacteriophage. In these experiments a second copy of the gIII modified to mimic biotin is inserted into the M13 multicloning site controlled by the induction of the IPTG-inducible lac promoter. This allows the suppression of the second copy of the surface protein in the absence of IPTG. Experiments to determine the presence of host microorganisms will be performed in the presence of IPTG, resulting in amplification of progeny displaying the unique epitope. A Salmonella detection assay is currently being developed by modifying bacteriophage P22 using a similar approach as described for M13. The displayed epitope for P22 is a 6xHis tag sequence consisting of 6 histidine residues, which will be inserted at the carboxy terminus of a second copy of the tail spike protein gene under control of the lac promoter. A Salmonella strain containing a functional lac repressor expressed at levels high enough to suppress the lac-controlled 6xHis tail spike protein must be used to generate the phage for the assay. The resultant phage will still be infectious but will only produce the tailspike protein upon infection of a wild type Salmonella. This platform potentially can be expanded to numerous pathogen-specific phages with different epitopes to allow detection of multiple pathogens in the same sample.
Related Documents:WM1, WM2, WM3, WM8
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Project 9 - Food Safety (Pathogen Detection)
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David Kotterman; email@example.com
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Internal Documents: Management: External Advisory
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