Creating Single-Copy Genetic Circuits and Microbial Kill Switches

Synthetic biology is increasingly used to develop sophisticated living devices for basic and applied research. Many of these genetic devices are engineered using multi-copy plasmids, but as the field progresses from proof-of-principle demonstrations to practical applications, it is important to develop single-copy synthetic modules that minimize consumption of cellular resources and can be stably maintained in a single-copy plasmid or as a genomic integrant. Here we use empirical design and iterative construction and testing to build single-copy, bistable toggle switches with improved performance and reduced metabolic load that can be stably integrated into the host genome. Based on the theory to create the single-copy circuit, we next develop biocontainment system that couples environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. This kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The synthetic gene circuits efficiently kill Escherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism. Overall, the design parameters developed and demonstrated here provide important guidance for future efforts to construct optimized single-copy circuits for practical applications.