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, such as bioremediation, wastewater pathogen detection, and nitrogen fixation for improved crop performance. However, deployed microbes have an inherent risk of negatively impacting balanced ecosystems, necessitating controlled release. 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 allow each released microbe to contain a built-in expiration date.. Our timer functions by utilizing removable, sequence-specific, transcriptional roadblocks called T7 RNA Polymerase (T7 RNAP) pause sites to partially stop the transcription of a T7 RNAP-Adenine Base Editor Fusion protein (the mutagenizer). Over days or weeks, the mutagenizer can mutate the sequence-specific pause sites, rendering them non-functional, which allows further downstream transcription and mutagenesis. We hypothesize that this shift in downstream transcription and mutagenesis can be coupled to a method to activate gene expression after a tunable delay period. At this point in the project, our prototype is effective at achieving our intended mechanism of delayed shifts in downstream transcription and mutagenesis and now we are investigating methods to couple this behavior to gene expression. For one approach, we are utilizing a RNA sensing technology called SNIPRs. SNIPRs are tools designed to be able to detect mutations in target RNA sequences called ‘triggers’, followed by expression of a user-defined protein payload in response. We hypothesize that SNIPRs can be utilized to detect specific mutations that appear over time through our timer mechanism, which can then be utilized to trigger payload expression. This summer we have been attempting to adapt the SNIPR sensor technology for our purposes and investigating important variables for SNIPR sensor performance, such as sensor expression strength and trigger sequence design. We are also designing a small library of sensor/ trigger pairs to screen for pairs that work well for activating gene expression in our timer system. Finally, we are investigating methods for achieving tunability in our timer system by making trigger mutations appear after different time intervals, such as 1,2, and 3 weeks.