Figure 5

Maps of next generation mini-Tn 5/7 - lux and mini-Tn 7 - lux delivery vectors. A) pTn5/7LuxK6. As in pTn5/7LuxK3 (Figure 2) the mini-Tn5/7-lux element is flanked by the Tn5 mosaic ends (ME) and carries a promoter-less P. luminescens luxCDABE operon, in this example the kanamycin resistance encoding nptII gene, the Tn7 left (Tn7L) and right (Tn7R) ends, the R6K origin of replication (ori _R6K), and an origin of conjugative transfer (oriT). The new generation of vectors is further functionalized by 1) transcription of the Tn5 transposase-encoding tnpA gene located outside of the transposable element by the constitutive S12 gene promoter (P _S12 ) from Burkholderia thailandensis; 2) flanking of the antibiotic resistance marker by functional Flp recombinase target (FRT) sites; and 3) flanking of the lux operon by attB sites for Gateway recombineering. The pTn5/7LuxG6, pTn5/7LuxT6 and pTn5/7LuxTc6 contain gentamicin, trimethoprim and tetracycline resistance markers, respectively. Other abbreviations are defined in the Figure 2 legend. B) pTn7oLuxK4. This vector contains many features of the pTn5/7Lux series but does not contain the Tn5 transposase gene or mosaic ends. Its unique features include two DraIII sites that because of the 3-nucleotide ambiguity (indicated by bold letters) in the DraIII recognition sites (DraIII-1 5′-CACTATGTG and DraIII-2 5′- CACCGCGTG) can be used for directional cloning of promoter-containing DNA fragments for transcription of the lux operon genes. In the illustrated example the lux operon is transcribed from the B. pseudomallei ompA promoter (P _ompA ). This is also indicated by the “o” in the plasmid name. The pTn7oLuxG4 and pTn7oLuxT4 contain P _ompA and the gentamicin (aacC1) and trimethoprim (dhfRII) resistance markers, respectively. Other abbreviations are as in A).