Development of a Tunable Genetic Fuse for Biocontainment

• Andrew Weissman, Chemical Engineering, University of Delaware

• Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware

Engineered microbes have a variety of exciting environmental applications including the potential for bioremediation and wastewater pathogen detection. However, deploying these technologies have an inherent risk of negatively impacting balanced ecosystems, meaning their release into the environment must be controlled. Currently, biocontainment strategies center around two approaches known as auxotrophy and inducible kill switches, but both are impractical when considering widespread environmental release of an engineered microbe. Our research project is developing a tunable biological timer that delays when a target gene, or “payload”, is expressed inside of a microbe. If the timer-activated gene encodes a “payload” that kills the cell, it could control the lifetime of microbial populations released into the environment, thus allowing them to accomplish their goal before dying. Our timer functions by utilizing transcriptional roadblocks called T7 RNA Polymerase (T7 RNAP) pause sites to partially stop T7 RNAP transcription. If the T7 RNAP makes it downstream of these pause sites, it can transcribe an actuator protein that triggers our payload’s expression. Each T7 RNAP is fused to an adenine base editor that can mutate roadblocks, breaking their ability to terminate transcription. Over time, this results in pause site removal, which eventually allows actuator transcription and system activation. Previous prototypes of the timer were “leaky” as T7 RNAPs were transcribing the actuator without any time delay. To solve this problem, we added more roadblocks and used a weaker T7 RNAP variant to more effectively inhibit T7 RNAP transcription. These approaches succeeded, but necessitated more efficient removal of the additional roadblocks. Thus, we explored whether fusing additional base editing enzymes and optimizing the pause site substrate could increase the frequency of pause site removal. Ultimately, these approaches led to a ~100X reduction in leakiness and a multi-fold improvement in pause site removal efficiency. This new prototype provides a greatly improved, delayed activation of the “payload”, potentially enabling proper timer function.