Date of Award
Master of Science (MS)
The utilization of genetically engineered microbes in environmental systems requires effective biocontainment mechanisms to ensure their safe operation. CRISPR-based kill switches are a promising solution, inducing cell death through site-specific DNA cleavage under specific environmental conditions. In this study, we aimed to assess the impact of CRISPR-based kill switches on the abundance and persistence of bacterial DNA. Our results revealed that while CRISPR-based kill switches were capable of achieving high reductions in viable Escherichia coli Nissle 1917 (EcN); the DNA largely remained intact. This was observed in two strains of EcN: one enabled with multi-locus genome cleavage, and the other with single-locus genome cleavage. The persistence of DNA in EcN was likely caused by the presence of multiple genome copies, which resulted in the creation of extra gene copies that were incompletely removed upon activation of the kill switch. Furthermore, we found that the DNA remained largely intracellular, and the cell membrane remained uncompromised, leading to a slow dissipation of DNA in surface water. The long persistence of DNA from some bacteria, particularly fast-replicating bacteria like EcN, may pose a risk to the accurate assessment of biocontainment using genetic markers and increase the likelihood of horizontal gene transfer of transgenic elements to other bacteria in the environment.
Dr. Kimberly Parker; Energy, Environmental & Chemical Engineering
Dr. Fangqiong Ling, Dr. Yinjie Tang