Host Evolution Improves Genetic Circuit Function in Complex Growth Environments

Genetically engineered bacteria have become an attractive platform for numerous biomedical and industrial applications. Despite genetic circuitry functioning predictably under favorable growth conditions in the lab, the same cannot be said when placed in more complex environments for eventual deployment. Here, we used a combination of evolutionary and rational engineering approaches to enhance E. coli for robust genetic circuit behavior in non-traditional growth environments. We utilized adaptive laboratory evolution (ALE) on E. coli MG1655 in a minimal media with a sole carbon source and saw improved dynamics of a population-lysis-based circuit after host evolution. Additionally, we improved lysis circuit tolerance of a more clinically relevant strain, the probiotic E. coli Nissle, using ALE of the host strain in a more complex media environment with added reactive oxygen species (ROS) stress. We observed improved recovery from circuit-induced lysis in the evolved Nissle strain, and in combination with directed mutagenesis, recovered circuit function in the complex media. These findings serve as a proof-of-concept that relevant strains of bacteria can be optimized for improved growth and performance in complex environments using ALE and that these changes can modify and improve synthetic gene circuit function for real-world applications.### Competing Interest StatementJ.H. is a co-founder of GenCirq Inc, which focus on cancer therapeutics. He is on the Board of Directors and has equity in GenCirq. His spouse is employed part time for the bookkeeping and to support employees with Human Resources.