BIOENGINEERING BACTERIOPHAGE FOR THE SENSITIVE & RAPID DETECTION OF BACTERIA IN WATER
Hinkley, Troy Cameron
Access to sanitary drinking water is a fundamental human right required for the continued prosperity of humanity. As a result, the World Health Organization (WHO) has published water safety recommendations stating that drinking water supplies should contain no detectable coliforms in 100 mL of water to be considered safe to drink. Finding a single bacterial colony forming unit in that volume is akin to finding a single grapefruit dropped into Lake Erie, a size difference of approximately 15 orders of magnitude. Only traditional culture techniques are capable of such detection limits, and all require at least 24 to 48 hours. The United Nations Children’s Fund (UNICEF) has declared the need for detection devices to accomplish the abovementioned detection limits in much shorter time frames. In response, we developed a series of bacteriophage-based biosensors with detection limits that approach and achieve the aforementioned detection requirements in a fraction of the time required for traditional culture techniques. Our most rapid assay is capable of detecting 10-20 CFU of E. coli in 100 mL of drinking water in only 5 hours. Our fully quantitative assay only takes 12 hours and is directly comparable to established methods. The sensitivity of our rapid bacteriophage-based detection technology is the result of multiple technologies leveraged together. We created novel reporter enzymes using genetic fusions between highly active enzymes and an affinity binding motif that irreversibly binds to cellulose. These constructs were inserted into phage genomes which served as sensitive biorecognition agents in drinking water detection assays. The novel chimeric reporters specifically bound to the cellulosic filters used in the detection assays, allowing for low detection limits in a fraction of the time required for standard methods.
Food science; food safety; Genetics; Bacterial Detection; Bacteriophage; Pathogen Detection; Phage; Water Safety; Microbiology
Nugen, Sam Rasmussen
Wiedmann, Martin; Peters, Joseph E.
Food Science and Technology
Ph. D., Food Science and Technology
Doctor of Philosophy
dissertation or thesis