Near-Perfect Digital Switching in a Synthetic Biosensor Circuit Achieved through Temporal Control of Circuit's Genetic Makeup

Near-perfect digital switching in a synthetic biosensor circuit achieved through temporal control of circuit's genetic makeup

Nicolas Lapique1 and Yaakov Benenson1*

1Department of Biosystems Science and Engineering, Swiss Institute of

Technology (ETH Zurich), Mattestrasse 26, Basel 4058 Switzerland

*To whom correspondence should be addressed: kobi.benenson@bsse.ethz.ch

ABSTRACT
Biosensing of endogenous molecular inputs is widely used in basic research and applications that range from bioproduction to medicine. Synthetic circuits applications can be safe and reliable only if a high sensitivity is achieved without sensor leakage in the Off state. High dynamic range with such sensors has only been achieved by exogenous ligand IPTG, but for endogenous microRNA inputs the range has been modest. Here we dramatically improve sensor dynamic range and virtually reduce leakage in the Off state to zero via temporal control of sensor genetic makeup. We engineer a delay in output gene availability relative to the repressor module using site-specific recombinase Cre or a cascade of Flp and Cre recombinases, and measure dynamic range of up to 2000-fold. A considerable reduction of the leakage is achieved without loss of sensitivity because the strategy does not increase the strength of the repressor module and therefore it does not result in concomitant decrease of the On. The near-perfect digital biosensor we engineer can lead to safe medical applications, enable reliable biosensing, and facilitate the construction of large synthetic gene networks.