Programming the Timing of Gene Expression in Synthetic Circuits

The control of gene expression timing in synthetic circuits has many potential applications for performing a target function using genetically modified cells including biocontainment by triggering cell death following a defined delay and controlling the timing of cargo release for drug delivery. Previous studies have demonstrated that bistable switches or transcriptional cascades can control the timing of gene expression. However, tuning the delay time requires sequence modifications and the length of the delay is correlated with output noise. To identify a robust and tunable delay generating circuit, we systematically enumerated a broad range of circuit topologies and scored circuit performance. We identified a molecular sequestration circuit that exhibits tunable control over the duration of the delay and does not significantly amplify output noise. This circuit establishes an adjustable threshold using a repressor to stoichiometrically inhibit an activator. The delay is determined by the time for the activator to accumulate to the level of the repressor. We experimentally characterized the range of delays for this circuit using heterologous extracytoplasmic function (ECF) σ and anti-σ factors in E.coli.  This delay generator circuit can be used as a building block for complex synthetic circuits including developmental programs and pulse generators.