Modified Tn transposon vectors for controlled chromosomal gene expression.

Complementation remains a foundation for demonstrating molecular Koch's postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal Tn site without the need for selection pressure. Here, we enhanced the utility of mini-Tn insertion vectors by the addition of inducible (P) and constitutive (PcL and P) promoters, allowing differential transcriptional control of genes integrated into the chromosome. We validated the utility of these promoters by cloning the gene, encoding green fluorescent protein, downstream of each promoter and integrating a mini-Tn construct harboring these elements into the Tn site on the chromosome of the K-12 strain MG1655. The PcL and P promoters provided equivalent levels of GFP expression and offered flexibility based on the target host strain. Activation of the tightly regulated P promoter with its inducer cumate resulted in tunable expression of GFP in a dose-dependent manner. We further demonstrated the tight control of the P promoter using the toxic genes, and the expression of which is detrimental to viability. Together, these modified mini-Tn vectors allowing differential control of genes integrated into the chromosome at a conserved site offer an efficient system for complementation where plasmid use is restricted.IMPORTANCEChromosomal integration using mini-Tn vectors provides an efficient means to insert genes into the chromosome of many gram-negative bacteria. Insertion occurs at a conserved site and allows for the stable integration of genes in single copy. While this system has multiple benefits for enabling complementation, a cornerstone for fulfilling molecular Koch's postulates, greater flexibility for controlled gene expression would enhance its utility. Here, we have added to the function of mini-Tn7 vectors by the addition of inducible and constitutive promoters and demonstrated their capacity to drive the controlled expression of target genes integrated into the chromosome. In addition to complementation, these modified vectors offer broad application for other approaches including chromosomal tagging, expression, metabolic engineering, and synthetic biology.