Digitalizing heterologous gene expression in Gram-negative bacteria with a portable on/off module

While prokaryotic promoters controlled by signal-responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an off state of zero transcription--as ideally needed for synthetic circuits. To overcome this problem we have modelled and implemented simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation-inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropylketone) and shown to perform effectively both in E. coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.