Synthetic biology has emerged as a promising field for addressing societal issues by utilizing genetically engineered (GE) organisms. However, practical applications often involve the release GE microbes (GEMs) into the environment. For example, while researchers have been developing GEMs to produce antimicrobial peptides in the gastrointestinal tract of livestock, providing new pathogen reduction strategies for farmers, these GEMs are expected to be released together with feces into the environment. Similarly, Rhodococcus opacus has been engineered for potential applications such as bioremediation of phenolic compounds and valorization of agriculture wastes, but these applications involve intentional or accidental release of GE R. opacus strains into the environment. Despite the promising application potentials of these GE microbes, the consequences of such releases are difficult to assess, and this biosafety concern should be carefully addressed.

In this work, we developed two CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917, a single-input chemical-responsive switch and a 2-input chemical- and temperature-responsive switch. Our results demonstrated that strains harboring either kill switch can be selectively and efficiently killed inside the murine gut, while strains harboring the 2-input switch are additionally killed upon excretion. Additionally, to develop and provide a generalizable “suicide” circuit in R. opacus that turns on only after the designed bioremediation “mission” is accomplished, we developed an Aromatic Passcode Kill Switch (APKS) by using a previously characterized aromatic responsive sensor and a killing module. This circuit allows R. opacus to perform phenolic compound degradation and self-destruct in a self-controlled and programmable manner by automatically sensing the changes in phenolic compound concentrations without any additional inducers or manual interventions. Our results show that the genetic kill switches can help maximize the potential benefits of synthetic biology while minimizing the potential risks of GEMs released into the environment.

Austin Rottinghaus

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