P. aeruginosa Mutant Library

Obtaining mutants from the Pseudomonas aeruginosa PAO1 transposon mutant library (Jacobs et al. 2003. PNAS 100, 14339)

Strains from the Seattle P. aeruginosa PAO1 transposon mutant library are available to the research community through a nonprofit cost center at the University of Washington. The accompanying table (“PA two-allele library”) lists the mutants available. The strains have been single colony-purified, and there are two mutants available for most nonessential genes. Both individual mutants and copies of the entire two-allele library are available. The first set of complete two-allele library copies has been distributed and we are accepting requests for the second set. When a sufficient number of requests has been received (8-10), the additional copies of the library will be generated and distributed.

Strains are available for a charge which covers preparation and shipping costs. To request a mutant, fill out the attached order form and send a copy of the original excel file to pamutant [ a t ] u.washington.edu. For requests from countries requiring import or other permits (e.g., Canada or Australia), the requestor must obtain and FAX copies to Kiara Held at 206-685-7301.

We can not accept credit cards as payment. The only acceptable payment methods are by Purchase Order number, check, or bank transfer.

After receiving a mutant, it is important that a sample of the strain be maintained as a frozen stock (–70°C) in the recipient laboratory. We recommend that the researcher streak from the stab onto a nutrient medium such as LB agar immediately after receipt, then scoop up a generous sample from the dense part of the plate for the frozen stock (in LB containing 5% DMSO v:v). Quality control is performed to ensure that you are sent a viable stock. It is possible that a strain you requested is viable for only a short period of time due to the mutation it harbors, and would not be recoverable after shipping. Once a strain has been shipped and is at that time viable, there will be no refunds or reshipment without an additional order. We also urge investigators to check the identities of mutants by PCR (or sequencing) prior to use, and that this information be shared with us for incorporation into the strain database. In quality control tests of the two-allele library, about 10% of mutant insertion sites did not match the original assignments. There are two steps to confirming a transposon insertion mutant: showing that the insertion location matches the prediction, and showing that the intact gene is absent. For each gene of interest, the researcher should design flanking primers or utilize the primers (designed by Mike Jacobs at the UWGC) listed in the table of strains (“ PA two-allele library ”). These primers should be tested initially using the wild type parent strain to verify production of an appropriate wild-type band. For insertion strains, the same PCR should yield either no band or a band corresponding to a very large product. Demonstrating the presence of the transposon insertion requires use of a transposon-specific primer. If the transposon used was ISphoA/hah, then use primer Hah minus 138 (5’cgggtgcagtaatatcgccct-3’), and if the transposon was ISlacZ/hah, use lacZ 148 (5’-gggtaacgccagggttttcc-3’). The transposon primer should be used in conjunction with one of the flanking primers, according to the orientation of the transposon relative to the gene. Additional information can be found at: http://www.genome.washington.edu/UWGC/pseudomonas/index.cfm .

When you have either positively or negatively confirmed a mutant strain, please send an email to pamutant [ a t ] u.washington.edu conveying the results.

The table of strains (“PA two-allele library”) carries information about each mutant in the two allele library. Most of the column headings are self-explanatory. The Location column provides internal identification and location information, the position in ORF column provides the transposon location and length of the ORF, the transposon direction (orientation) relative to the genome is provided, the frame column provides the reading frame of the transposon relative to the target gene (with +2 corresponding to in-frame lacZ or phoA), the sequences and locations of pairs (forward and reverse) of primers for confirmation of transposon locations are given, as are the lengths of wild-type fragments amplified. The “Confirmed?” column identifies insertions which have been confirmed (or not confirmed) by our or other groups. In cases in which sequence analysis placed insertions at novel sites in the genome, the ORF affected is identified. We regard these novel insertion sites as tentative.

Mutant Order Form (excel spreadsheet)

PA two-allele library (excel spreadsheet)