From Sequence to Cell to Population: Secure and Robust Biosystems Design for Environmental Microorganisms

Genetically engineered microorganisms (GEMs) hold significant promise for use in a variety of human health and environmental applications, but the lack of robust and generalizable biocontainment strategies currently hinders technology adoption and public trust. To reduce the risk of unintended consequences from deployed GEMs, built-in security mechanisms are needed to ensure that GEMs function where and when needed without proliferating beyond target conditions. The overarching goal of the Secure Biosystems Design Scientific Focus Area (SFA) at LLNL is to develop robust, generalizable biocontainment strategies in environmentally relevant soil microbes (e.g., Pseudomonas species) at the sequence, cellular, and population levels. At the sequence level, we are currently using high-performance computing (HPC) and high-throughput synthesis and screening strategies to advance a synthetic gene entanglement concept for containment. Here, two genes are encoded within different coding frames of the same sequence space to protect engineered functions against mutational inactivation and/or to mitigate the horizontal transfer of potentially invasive genes. Building on this layer of sequence stability, we are also developing sense-and-respond circuits that constrain the survival and function of plant-benefiting microorganisms to their target application environments. Here, our current work is focused on identification of toxin systems and essential genes that can be used for kill-switch and synthetic auxotrophy functions, respectively, in Pseudomonas species. Control strategies, including multi-signal integration, pseudotaxis, and quorum sensing-based population coordination, will eventually be incorporated to increase the overall system robustness to environmental fluctuations. Ultimately, our work will yield safeguard mechanisms that control the niche-specific function of GEMs and prevent the transfer of potentially “invasive traits” to neighboring native microorganisms, thereby enabling safer and more effective use of GEMs in environmental applications.

Work at LLNL is performed under the auspices of the U.S. Department of Energy at Lawrence Livermore National Laboratory under Contract DE-AC52- 07NA27344 (LLNL-ABS-822649).