Influence of Environmental Conditions on the Escape Rates of Biocontained Genetically Engineered Microbes

The development of genetically engineered microbes (GEMs) has resulted in an urgent need to control their persistence in the environment. The use of biocontainment such as kill switches is a critical approach to prevent the unintended proliferation of GEMs; however, the effectiveness of kill switches─reported as escape rates, i.e., the ratio of the number of viable microbes when the kill switch is triggered relative to the number when it is not triggered─is typically assessed under laboratory conditions that do not resemble environmental conditions under which biocontainment must perform. In this study, we discovered that the escape rate of an <i>Escherichia coli</i> GEM biocontained with a CRISPR-based kill switch triggered by anhydrotetracycline (aTc) increased by 3-4 orders of magnitude when deployed in natural surface waters as compared to rich laboratory media. We identified that environmental conditions (e.g., pH, nutrient levels) may contribute to elevated escape rates in multiple ways, including by altering the chemical speciation of the kill switch trigger to reduce its uptake and providing limited nutrients required for the kill switch to function. Our study demonstrated that conditions in the intended environment must be considered in order to design effective GEM biocontainment strategies.