Digital switching in a biosensor circuit via programmable timing of gene availability

Transient delivery of gene circuits is required in many potential applications of synthetic biology, yet pre-steady-state processes that dominate this delivery route pose significant challenges for robust circuit deployment. Here we show that site-specific recombinases can rectify undesired effects by programmable timing of gene availability in multi-gene circuits. We exemplify the concept with a proportional sensor for endogenous microRNA and show dramatic reduction in its ground state leakage thanks to desynchronization of circuit’s repressor components and their repression target. The new sensors display dynamic range of up to 1000-fold compared to 20-fold in the standard configuration. We applied the approach to classify cell types based on miRNA expression profile and measured >200-fold output differential between positively- and negatively-identified cells. We also showed major improvement of specificity with cytotoxic output. Our study opens new venues in gene circuit design via judicious temporal control of circuits’ genetic makeup.