Figure 1. Construction of an E. coli host strain and a genomic library of P. aeruginosa.

A) A fabA′-′lacZY translational fusion was assembled on a mini-Tn7 suicide delivery vector. B) The mini-Tn7 vector was co-electroporated with the Tn7 transposase expressing helper plasmid pTNS1 into an E. coli Δlac strain. Since the suicide delivery vector cannot replicate in E. coli due to the presence of the conditional protein-dependent oriR6K, gentamycin-resistant (Gm^r) transformants will result from site- and orientation-specific integration at the chromosomal attTn7 site which is located immediately downstream of the glmS gene in the glmS - pstS intergenic region. C) A PstI-EcoRI P. aeruginosa chromosomal DNA library was constructed by ligation of partially digested PstI-EcoRI fragments into pUC18. D) The library was used to transform a P. aeruginosa strain harboring a chromosomally integrated fabA′-′lacZY fusion. Since fabA′-′lacZY is only expressed at low levels, the host strain will only form light blue colonies on X-Gal-containing indicator medium. Transformants expressing putative activating proteins indicated by “+” will appear as darker blue colonies. Abbreviations: Ap^r, ampicillin resistance; FRT, Flp-recombinase target; Tn7L and Tn7R, left and right end of Tn7, respectively.