Engineering bioremediating bacteria for open-release application
Citation:
Svoboda, J. AND J. Reichman. Engineering bioremediating bacteria for open-release application. 2024 Synthetic Biology: Engineering, Evolution & Design (SEED) Conference, Atlanta, GA, June 24 - 27, 2024.
Impact/Purpose:
This presentation will be made at the 2024 Synthetic Biology: Engineering, Evolution & Design Conference, and it covers ground-breaking research at ORD/CPHEA/PESD within Product CSS.403.2.1 - Data to support risk assessments of novel engineered microbes. The poster will showcase plans for bioengineering of an Escherichia coli strain with synthetic constructs for remediation of a prevalent microplastic pollutant (PET) and for biocontainment via a CRISPR-Cas12a kill-switch. The research will develop a model chassis organism to be used in experiments to address the Agency needs for updated detection and tracking methods for genetically engineered microorganisms (GEMs) being tested under containment for their persistence, performance, and efficacy.
Description:
From Superfund sites, to oil spills, to the ubiquity of microplastics, anthropogenic pollution is an ever-growing threat to human health and the environment. Synthetic biology can potentially address this problem with microbes engineered to degrade pollutants, and also further improve such systems with biocontainment methods to prevent undesired spread of the microbe. However, there are uncertainties regarding the safety of engineered microbes with synthetic constructs intended for open release into the environment. To determine the stability of such engineered bacteria and their effects on native microbiota, we aim to engineer a strain of Escherichia coli that will die upon completing its bioremediation task, in order to test its functional efficiency for both bioremediation and biocontainment. Focusing on microplastic as our example bioremediation target, we chose poly(ethylene terephthalate) (PET) as it is widely-used, including in consumer products such as water bottles. Using a CRISPR-Cas12a recombineering system, PET-degrading enzymes will be integrated into the genome, along with a kill-switch circuit. The kill-switch consists of Cas12a and a CRISPR array with spacers targeting multiple locations throughout the E. coli genome, including the introduced PET-degrading enzyme to prevent spread of the synthetic genetic construct. Kill-switch expression is modulated by a genetic circuit designed to induce cell death upon the absence of the environmentally benign PET degradation product. Once assembled, this strain will be grown in both synthetic media and soil microcosms, assessing PET degradation and loss of cell viability after PET has been fully degraded. We plan to track the integrity and expression of the synthetic PETase construct over time. In the microcosms, we can further leverage metagenomics and transcriptomics to observe the effects of the engineered strain on the native soil microorganisms. This work will give insight into the interplay between synthetic gene constructs, engineered microbes, and the native microbiome in open-release bioremediation, allowing both researchers and regulators to make more informed decisions.