Developing Microbial Biocontainment Strategies and Their Assessment Methods

EPA Grant Number: R840205
Title: Developing Microbial Biocontainment Strategies and Their Assessment Methods
Investigators: Moon, Tae Seok , Parker, Kimberly M
Institution: Washington University in St. Louis
EPA Project Officer: Callan, Richard
Project Period: July 1, 2021 through June 30, 2024
Project Amount: $744,262
RFA: Assessment Tools for Biotechnology Products (2020) RFA Text |  Recipients Lists
Research Category: Chemical Safety for Sustainability , Safer Chemicals

Objective:

With advances in synthetic biology, development of genetically engineered microbes (GEMs) for environmental applications has been accelerated. One critical concern with the use of GEMs in the environment is their spread after the intended mission is accomplished. Genetically-programmed biocontainment strategies (e.g., kill-switches) have been developed to control the viability of GEMs. However, multiple major challenges must be overcome to implement them for real-world applications. First, kill-switches must respond to application-relevant signals (e.g., pollutants) instead of laboratory chemicals conventionally used for kill-switch development. Second, studies investigating the efficacy and mutational long-term stability of kill-switches in environmental media are extremely limited, and assessment methods to evaluate biocontainment strategies must be developed considering application-relevant environmental conditions. Third, the regulatory criterion for biocontainment must be met, and the assessment systems must ensure a close tie between lab observation and field prediction of biocontainment efficacy and stability. To this end, we will develop new approaches to assess the efficacy and long-term stability of biocontainment under application-relevant environmental conditions. We will also create a kill-switch that enables “suicide” after GEMs degrade target pollutants, demonstrating both bioremediation and induced GEM killing in simulation scenarios.

Approach:

In this project, we will advance stepwise from our currently available lab chemical-based kill-switches in Escherichia coli to a pollutant-based kill-switch in Pseudomonas putida applicable to bioremediation. First, we will develop and validate methods to quantify GEMs in soil and surface water. In addition to traditional colony counting, we will optimize our sensitive and field-applicable, quantitative PCR-based method for selective detection of GEMs. We will apply these techniques to measure escape rates in microcosm experiments and to assess inhibition of the kill-switch by dissolved constituents. Furthermore, we will test the hypothesis that environmental stress will increase the mutation rate, leading to an increase in the escape rate. Second, we will develop kill-switches in new strains of E. coli to test the hypothesis that deletion of genes associated with error-prone repair processes will decrease the escape rate and increase the long-term biocontainment stability. Third, we will develop a pollutant-dependent kill-switch in P. putida and assess its performance in remediation simulation scenarios. 

Expected Results:

This project will provide a validated assessment system to quantify GEM abundance in environmental media and to determine the susceptibility of biocontainment strategies to inhibition and mutation. This system will serve as the first checkpoint before considering field tests that involve GEMs containing genetic biocontainment systems. Additionally, this project will provide a novel remediation-relevant kill switch that will become a benchmark biocontainment strategy. Thus, this project will provide technologies that minimize the risks associated with environmental applications of GEMs to ensure public safety.

Supplemental Keywords:

genetically modified organism; biosafety; cleanup; genetic circuit