FIG. 1.

Genetic elements used in the expression system. (A) The regulatory module contains the mini-Tn5 insertion sequences flanking a kanamycin resistance gene to select for transposition and a nahR-Psal regulatory system controlling the expression of xylS2. The expression modules constructed in this study are two plasmids, including pTSPm, a mini-Tn5 delivery vector that contains the Tn5 insertion sequences flanking a spectinomycin-streptomycin interposon, the Pm promoter, and a unique NotI site where a gene of interest may be cloned by using auxiliary plasmids (8). (B) pCCD5, a ColE1 vector containing a transcriptional terminator, Pm, and an MCS with an efficient translation initiation region, which are flanked by NotI sites. This plasmid allows subcloning of NotI fragments with Pm fusions to heterologous genes to the unique sites of mini-Tn5 delivery systems. RBS, ribosome binding site.