DEVELOPMENT OF KILL-SWITCHES FOR BIOCONTAINMENT OF GENETICALLY ENGINEERED MICROBES

Synthetic biology is a promising research field for many practical applications, involving use of genetically engineered (GE) microorganisms that are often expected to be released into the environment. For example, scientists have been developing GE probiotics to produce antimicrobial peptides in the gastrointestinal tract of livestock, providing new pathogen reduction strategies for farmers, but administered GE probiotics are expected to be released together with feces into the environment. Similarly, Rhodococcus opacus has been engineered for potential applications such as bioremediation of contaminated sites and valorization of agriculture wastes, but these applications involve intentional or accidental release of GE R. opacus strains into the environment. Despite the potential of these GE microbes, the consequences of such releases are difficult to assess, preventing federal regulatory agencies from approving those potential applications.The long-term goal of this project is to develop a generalizable biocontainment circuit that can be used in a variety of application-relevant GE microbes. Specifically, we aim to develop and provide a generalizable "suicide" circuit that turns on only after the designed "mission" is accomplished. As the first step towards achieving this long-term goal, we propose two specific objectives.[1] We will demonstrate "suicide" circuits in GE Escherichia coli Nissle (EcN) that will be engineered to produce antimicrobial peptides. This probiotic EcN strain can be potentially used as a "pathogen killer" in the animal gut, which commits "suicide" when released into the environment.[2] We will develop "suicide" circuits in a consortium of GE R. opacus strains that will be engineered to consume toxic aromatic compounds. This consortium of GE R. opacus strains can be potentially used as pollutant degraders in a contaminated site, which commit "suicide" when the cleanup is completed.