Abstract
The rational design of synthetic regulatory circuits critically hinges on the availability of orthogonal and well-characterized building blocks. Here, we focus on extracytoplasmic function (ECF) σ factors, which are the largest group of alternative σ factors and hold extensive potential as synthetic orthogonal regulators. By assembling multiple ECF σ factors into regulatory cascades of varying length, we benchmark the scalability of the approach, showing that these 'autonomous timer circuits' feature a tuneable time delay between inducer addition and target gene activation. The implementation of similar timers in Escherichia coli and Bacillus subtilis shows strikingly convergent circuit behavior, which can be rationalized by a computational model. These findings not only reveal ECF σ factors as powerful building blocks for a rational, multi-layered circuit design, but also suggest that ECF σ factors are universally applicable as orthogonal regulators in a variety of bacterial species.