Highly Modular “Bow-Tie” Gene Circuits with Programmable Dynamic Behavior

Despite synthetic biologists' best efforts, many synthetic gene circuits operate only when their components â?? the controlling inputs, the input-processing module, and the outputs â?? are extensively mutually optimized. Implementing flexible, programmable tools to control and reprogram living systems is still difficult. A possible solution lies in the application of the "bow-tie" network architecture. This architecture stipulates a focal component - a "knot" - mediating between the input processing module and the outputs, simplifying swapping/adding components, and introducing additional layer of control. Here we show synthetic bow-tie circuits that transduce multiple microRNA inputs into multiple protein outputs in cultured human cells, such that both the circuit logic and output dynamics are set independently and in parallel. Output dynamics are adjusted via two different knot configurations: the first being a transcriptional activator resulting in outputs reversibly tracking changes in the inputs; and the second comprising a recombinase-based cascade, transducing transient inputs into irreversible actuation. We experimentally constructed and extensively characterized bow-tie circuits in HEK293 cells, confirming their modularity, flexibility and scalability. We also validated the system using endogenously-expressed miRNA as inputs in additional cell lines. This novel platform can be used for biotechnological and biomedical applications in cell culture, model organisms and potentially in human therapy.