A modular chromosomally integrated toolkit for ectopic gene expression inVibrio cholerae

AbstractThe ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression inVibrio cholerae. We compare the broadly used promoter elements Ptacand PBADto versions that have an additional theophylline-responsive riboswitch (Ptac-riboswitch and PBAD-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with Ptac> PBAD> PBAD-riboswitch > Ptac-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for Ptac> Ptac-riboswitch > PBAD; while the newly developed PBAD-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible PBAD-riboswitch construct using the dynamic activity of type IV competence pili inV. choleraeas a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study ofVibrio choleraeand could be adapted for use in other species.