Refactoring two-component systems for tunable gene expression regulation and upgraded bacterial sensing.

Two-component systems (TCSs) are ubiquitous multi-step signal sensing systems in prokaryotes and are promising platforms for building cellular sensors. However, their programmability remains underexplored, limiting broader applications in synthetic biology. Here, we refactor TCSs to systematically elucidate the functional properties of response regulator (RR) and histidine kinase (HK) as the concentration-dependent activator and inhibitor for TCS sensor output, respectively. By decoupling HK expression from native feedback circuitry, we engineer ultrasensitive TCS sensors with tunable detection thresholds. By leveraging RR as a transducer, we couple one-component system (OCS) and TCS to create a synergistic sensing system (SSS) characterized by both a low detection limit and a high dynamic range. We further show that RR alone serves as a biological-low noise amplifier (LNA), substantially upgrading performance of diverse genetically encoded biosensors. Our study demonstrates TCS's high plasticity and programmability for customizing gene expression regulation in synthetic circuits, providing modular toolkits for biosensor optimization. A record of this paper's transparent peer review process is included in the supplemental information.