Genetic "expiry-date" circuits control lifespan of synthetic scavenger bacteria for safe bioremediation.

Synthetic biology enabled the systematic engineering of bacteria for diverse applications, but their deployment in open environments raises concerns about their persistence and unintended ecological impacts. To address these challenges, genetic "expiry-date" circuits were designed to impose a tunable lifespan on bacteria. These circuits, structured as a feedforward activation network, regulate the timing of cell death by controlling the expression of Lysis E, enabling a programmed lifespan ranging from hours to days. The lifespan can be tailored by modifying the number of activation steps in the cascade. The circuits were optimized by reducing gene expression leakiness of Lysis E using a synthetic small regulatory RNA and combining it with an asd-based auxotrophic system. The bacteria harboring the "expiry-date" circuits resulted in a GMO escape rate below U.S. NIH release standards (<10-10). To validate the practical applicability of this system, a synthetic phenol-scavenging Escherichia coli was constructed, which possessed enhanced phenol tolerance and phenol-detoxification capability, and harbored the "expiry-date" circuits. The engineered bacteria detoxified 0.1 g/kg of phenol in soil within 4 days and self-destructed by day 5. These results support the circuit's potential as a biocontainment strategy for the safe and controlled deployment of synthetic bacteria in real-world applications.