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, non-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 a 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/dicyclopropyl ketone) and shown to perform effectively in both Escherichia coli and the soil bacterium Pseudomonas putida.

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