Figure 2
Methods for site-specific insertion of promoter- lux fusions using mini-Tn 7-lux elements. Chromosomal integration of promoter-lux fusions can be achieved in two ways. (A ) The plasmid recovered in step 3 of the promoter identification and capture procedure illustrated in Figure 1 is a functional mini-Tn7 delivery plasmid which in some instances (e.g. when the plasmid contains short regions of promoter-containing chromosomal DNA or when RecA-deficient target strains are available) may be used to directly transpose site-specifically into the glmS gene-associated Tn7 attachment site (attTn7) in the chromosome of the bacterium under study to obtain luminescent derivatives. Site-specific mini-Tn7-lux insertion is achieved by co-transfer of the mini-Tn7-lux delivery plasmid and a helper plasmid that encodes the site-specific TnsABCD transposition pathway, which acts on the Tn7 left (Tn7L) and right (Tn7R) ends. Both plasmids contain the origin of transfer (oriT) for conjugal transfer into the target bacterium and the conditional R6K origin of replication (ori R6K), which limits their replication to E. coli hosts expressing the plasmid R6K π protein. The mini-Tn5 element (sequences flanked my the mosaic ends, ME) contained on the mini-Tn7-lux delivery plasmid does not transpose because the delivery plasmid does not encode Tn5 transposase. (B ) Alternatively, the promoter identified by sequencing the mini-Tn5/7-lux-chromosomal junction sequences located on the plasmid rescued in step 3 of the procedure illustrated in Figure 1 can be cloned into other mini-Tn7-lux elements. These are then transposed into the target bacterium for obtaining bioluminescent bacteria by site-specific mini-Tn7-lux transposition as described above. In both scenarios, A and B, the Kmr selection marker contained on the chromosomally integrated mini-Tn7-lux elements is flanked by Flp recombinase target (FRT) sites for optional marker excision with Saccharomyces cerevisiae Flp recombinase. The ampicillin resistance (Apr) marker is used for selection and maintenance of the helper plasmid in E. coli.