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Broad host-range mini-Tn7 vectors facilitate integration of single-copy genes into bacterial chromosomes at a neutral, naturally evolved site. Here we present a protocol for employing the mini-Tn7 system in bacteria with single attTn7 sites, using the example Pseudomonas aeruginosa. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. aeruginosa by either transformation or conjugation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. From start to verification of the insertion events, the procedure takes as little as 4 d and is very efficient, yielding several thousand transformants per microgram of input DNA or conjugation mixture. In contrast to existing chromosome integration systems, which are mostly based on species-specific phage or more-or-less randomly integrating transposons, the mini-Tn7 system is characterized by its ready adaptability to various bacterial hosts, its site specificity and its efficiency. Vectors have been developed for gene complementation, construction of gene fusions, regulated gene expression and reporter gene tagging.

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For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.

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