Obtaining mutants from the Pseudomonas aeruginosa PAO1 transposon mutant library (Jacobs et al. 2003. PNAS 100, 14339; Held et al 2012. J. Bacteriol., 194, 6387-6389)
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.
Strains are available for a charge. To request a mutant, fill out the order form that corresponds to your type of institution (academic institution order form, nonacademic institution order form, it may work best using Chrome, Internet Explorer, or Firefox) 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 email pamutant [ a t ] u.washington.edu (preferred) or fax copies to Kiara Held at 206-685-7301.
We are able to accept payment by purchase order number, check, bank transfer, or by using a credit card via a telephone transaction for small orders; however, we require a PO be emailed with your order form for orders >$400.
Maintaining Strains: After receiving a mutant, it is important that a sample of the strain be maintained as a frozen stock (–80°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.
Reconfirming Strains: We also urge investigators to check the identities of mutants by PCR (or sequencing) prior to use. We recommend that researchers test at least 10 isolated colonies by PCR for each strain as while we have made every attempt to single colony purify, due to the nature of high throughput, some cultures are mixed. 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. There is additional information about this procedure in the additional information link at the bottom of this page.
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, and there is also an Excel sheet with explanations. The primer sequences are suggestions only, and they have not been tested. If we have obtained two or more Sanger sequencing reads corresponding to within 500bp of the called insertion site, there is a + in the Sanger confirmed column. If we were able to detect the exact sequence of the called insertion site via Tn-seq performed on a pool of the entire library, there is a + in the Tn-seq confirmed column. If we were able to detect an insertion site within 100bp of the called insertion site via Tn-seq of the library pool, and the Sanger read that was used to call the location was not an exact read there is a (+) in the Tn-seq confirmed column. Basically, Tn-seq confirmations suggest the transposon insertion site exists in the library but it does not say it is in the assigned well, whereas Sanger sequencing is done on specific wells. We therefore believe our highest quality mutants to be confirmed by both Sanger and Tn-seq.
When ordering strains from the PA two-allele library, please include at least the “Location” and the “PAORF” from the “PA Two-Allele Library” Excel sheet on your order form. An example of this is… lacZbp01q1A01 PA1124.
Replacements: In creating a large arrayed mutant library like this one, it is inevitable that some assignments will fail to check out. We have done our best to minimize cross-contamination and insertion mis-assignment by colony purification and two rounds of sequencing. We have included multiple independent insertion mutants for most genes, and suggest that multiple mutants corresponding to genes of interest be requested to help provide coverage in case individual mutants can’t be confirmed. Unfortunately, we are unable to provide replacements for mutants that cannot be confirmed.
Mutant Naming: In publications, please reference strains from the Two-Allele Library by strain name (PW####) and refer to the genotype in the following way: gene name (or PAORF if there is no gene name)-well name (final three digits of the location field) as the allele number ::Transposon name (ISphoA/hah or ISlacZ/hah). For example, for strain PW1001, the genotype is recF-B10::ISphoA/hah, and for strain PW1003, the genotype is PA0005-E05::ISlacZ/hah. Please acknowledge grant # NIH P30 DK089507 on publications resulting from the use of these strains.
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