Improving a Tn7-based luciferase reporter system for promoter activity studies.

Single-copy chromosomal integration systems are essential tools for stable gene expression in bacteria, minimizing variability associated with plasmid-based systems. The Tn7 transposon-based system is widely used for this purpose, and one important application is the generation of reporter systems, such as the bioluminescent luxCDABE operon (lux). However, current Tn7-lux vectors exhibit undesirable background expression due to cryptic promoter activity near the antibiotic resistance cassette. Here, we report the construction of an improved vector, pTn7-lux-B0015, incorporating a strong synthetic terminator upstream of the lux operon. This modification effectively eliminated basal luminescence in the absence of a promoter and enhanced the dynamic range and responsiveness of the reporter. Using a Xanthomonas citri type III secretion system promoter as a model, we demonstrate that pTn7-lux-B0015 enables more accurate detection of gene expression under relevant growth conditions. This vector provides a valuable tool for the development of precise and tunable bioluminescent reporters in bacterial systems.