Approximately 34,000 mutants comprise the library as of February 2006(17). The mutants have been subjected to arbitrary PCR and sequencing to locate each transposon insertion site. The distribution of hits is shown in Figure 1 Each predicted PA14 essential gene has been hit an average of 4 times. To date, 1367 homologs of P. aeruginosa strain PA01 genes have not been hit. We are in the process of creating and analyzing at least 6,000 more PA14 mutants in the future as a part of a project funded by the Cystic Fibrosis Foundation.

The majority of the mutants have been constructed using a mariner-based transposon, MAR2xT7(Figure 2), developed in our laboratory (MAR2xT7 Construction). MAR2xT7 contains a gentamycin resistance cassette and outwardly-directed T7 promoters with short (28 bp) inverted repeats at each end, facilitating TraSH (Transposon Site Hybridization) analysis of mixed mutant populations (9 and 14).

While running pilot experiments, several PA14 mutants were created with the TnPhoA transposon (1). In addition, small subsets of the library mutants were made in two different exoU mutant backgrounds (Genetic Backgrounds). Mutants are available to academic researchers upon request for a nominal fee to reimburse us for out-of-pocket expenses(Request Mutant). Commercial scientists interested in requesting mutants should contact Dr. Frederick M. Ausubel.