<p>The major goal of this project is to develop a fully autonomous autobioluminescent yeast strain for use as a low cost, high-throughput tier 1 screening tool capable of reporting the presence of dioxins and dioxin-like chemicals in the U.S. food supply for improved assurance of food safety.
<br>The objectives for achieving this goal include:
<li>Develop a fully autonomous yeast-based dioxin bioreporter based on the bioluminescent bacterial luciferase gene cassetteThis objective will leverage traditional and synthetic gene assembly techniques to optimize a bacterial luciferase gene cassette for expression in a yeast-based host. The successful completion of this objective will yield a novel yeast strain capable of sensing and reporting the presence and bioavailability of a wide variety of dioxin and dioxin-like compounds in food and feed commodities.</li>
<li>Characterize the effectiveness of the dioxin-responsive yeast bioreporterThis objective will evaluate the effectiveness of the yeast bioreporter for dioxin detection and monitoring. The yeast bioreporter will be challenged with known concentrations of dioxin and dioxin-like compounds to determine minimum detection times, limits of detection, EC50 values, and relative effect potency.</li>
<li>Competitively evaluate the performance of the dioxin-responsive yeast bioreporter with currently available alternative detection assays. This objective will compare the yeast bioreporter assay against alternative dioxin screening assays in order to provide a competitive cost/benefit analysis and highlight the relative advantages and weaknesses of each assay under both laboratory and agriculturally relevant screening scenarios.</li></ol></p>
<p>The methods for the project will apply synthetic biology techniques to enable a set of prokaryotic bacterial genes to be expressed in a eukaryotic yeast host cell. The bacterial genes being used will confer a bioluminescent phenotype to the yeast cells, thereby allowing the yeast cells to function as bioreporters capable of surveying their environment for user-specified chemical or physical agents and responding via the generation of a bioluminescent light signal. In this particular research effort, we have chosen dioxins as the chemicals of choice because current cell-based assays for dioxins are time consuming and expensive. The development of a faster and cheaper yeast-based bioluminescent assay for dioxin detection and monitoring will enhance consumer food and animal feed safety. The measurement of success of our project goals will rely upon evaluations that include the collection of data pertinent to understanding how quickly the assay responds to dioxins, the lowest dioxin concentrations capable of being detected, and the potency or half maximal effective concentrations (EC50). Validation of success will then be obtained by comparing the yeast bioreporter assay to existing assays that are available in the marketplace. Key milestones will be a yeast bioreporter assay capable of detecting dioxins within the regulatory range established by the World Health Organization (0.4 to 4.0 parts-per-trillion) that can be completed in several hours rather than the multiple days required using existing assays. An additional milestone aims to reduce the cost of the assay over existing assays by 50%. Efforts to deliver the knowledge gained from our studies will be pursued through publication in the scientific literature and presentations at conferences focused on food safety.</p>