Manoil Lab

MUTANT DISTRIBUTION IS BEING DISCONTINUED. Individual mutant and pooled mutant library distribution will end October 15, 2022 and we will stop accepting new orders September 15, 2022. We will continue to distribute our few remaining complete single mutant arrayed libraries for P. aeruginosaA. baumannii and K. pneumoniae until December 15, 2022 or until our copies of them are exhausted.  

 

P. aeruginosa Mutant Library

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 arrayed P. aeruginosa PAO1 Two-Allele Transposon Mutant Library are available for a charge to the research community through a nonprofit cost center at the University of Washington. Complete copies of the library and transposon mutant pools are also available. The strains have been single colony-purified, and there are two mutants available for most nonessential genes. In-depth information about the library can be found in the following publications: Jacobs et al. 2003, PNAS 100:14339 and Held et al. 2012, J. Bacteriol. 194:6387. The accompanying Excel file (PA two-allele library) lists the mutants available.

Requesting individual mutants  

The library Excel file (which can be downloaded from the link below) provides information about each mutant in the two-allele library.  Most of the column headings are self-explanatory.  There is also a description of column headings on the second sheet of the file (“Legend”).  We apologize, but as a small research lab, we cannot send out replacements if our transposon location in a mutant cannot be confirmed or has been mis-assigned.  We do appreciate knowing confirmation status and will update our library file with the results as an aid to future users.

To request individual mutants, fill out the order form that corresponds to your type of institution (either academic or nonacademic, links at the bottom of this page) and send the completed Excel file to pamutant [ a t ] uw.edu. There is an instruction sheet in the order form, but please let us know if you have any questions about the form itself, the mutants, or the ordering process.

For requests from countries requiring import or other permits, the requestor must obtain the necessary permits and email copies to pamutant [at] uw.edu.  All charges resulting from failure to provide the required permits will be paid by the requestor, including the cost of return shipment following customs rejection.  While email is preferred, copies of permits may be faxed to the Manoil Lab at +1-206-685-7301.

We are able to accept payment by purchase order number, check, bank transfer, or credit card for most orders.  Credit card payments may be made via a secure website; the link to the website will be provided with the invoice.  We require a purchase order to be emailed with the order form for orders of $1,000 or more.

Choice of strains

In creating a large arrayed mutant library like this one, it is inevitable that some assignments will fail to check out. In addition, high-throughput growth and distribution may lead to some mixed cultures.

We have done our best to minimize cross-contamination and insertion mis-assignment by colony purification and two rounds of sequencing of insertion location (but not absence of the corresponding wild-type gene).

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 insertion locations to be those confirmed by both Sanger and Tn-seq. 

We have included more than one insertion for most genes, and suggest that multiple mutants corresponding to genes of interest be requested to help provide coverage in case individual mutants cannot be confirmed. Unfortunately, we are unable to provide replacements for mutants that cannot be confirmed.

Receipt and maintenance of strains

Individual strains are sent as stab cultures in semisolid agar.  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 (without antibiotic) immediately after receipt, then scoop up a generous sample from the dense part of the streak 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.

Confirmation of 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 of each strain by PCR both with flanking primers and with a transposon-specific primer paired with a flanking primer to confirm mutants.
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 Excel library file (“PA two-allele library”). The primers are computer-generated and have not been tested by us. They 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.

To demonstrate the presence of the transposon insertion, use a transposon-specific primer with one of the flanking primers.  The transposon used in each strain is listed in column K of the library Excel file.  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 “Strain confirmation procedures” link at the bottom of this page.

On occasion, purified transposon mutant strains produce both wild-type and insertion mutant bands in the PCRs described above. We suspect these are usually bacteria carrying both wild-type and insertion mutant alleles and arise from transposon insertion in one copy of a tandem duplication that includes the gene of interest. Such strains are frequently found for essential genes in which wild-type function is required for viability.
When you have either confirmed or been unable to confirm a mutant strain, please send an email to pamutant [at] uw.edu to let us know your results.

Strain variation due to mexT mutations

There are variations in the mexT gene commonly seen for PAO1 strains, including MPAO1 and the mutants in our transposon mutant library. The variations arise because MPAO1 and mutants of it carry an inactivating mutation in the mexS gene, which leads to constitutive activity of the MexT regulator. The constitutive MexT activity slows growth and reduces survival of frozen strains, and (different) mexT-minus mutations can thus be enriched in frozen stocks of mutants or during strain passaging. The mexT- minus mutants can be distinguished from mexT-plus strains because they form somewhat larger colonies and are more sensitive to chloramphenicol (tested on LB agar containing 10 micrograms/mL chloramphenicol) and nalidixic acid (tested on LB supplemented with 64 micrograms/mL nalidixic acid). Two papers describing the variation are: Luong PM, et al. Emergence of the P2 Phenotype in Pseudomonas aeruginosa PAO1 Strains Involves Various Mutations in mexT or mexF. Journal of Bacteriology. 2014;196(2):504-513. doi:10.1128/JB.01050-13, and Lee et al, Reconstructing a wild-type Pseudomonas aeruginosa reference strain PAO1. Journal of Bacteriology. 2021; 203(14):e00179-21 doi:10.1128/JB.00179-21.

Pooled transposon mutant library

Our P. aeruginosa transposon mutant pooled library was made by saturation-level transposition in MPAO1.  To achieve high insertion density, we used a transposon with low insertion specificity derived from Tn5.  To limit false assignments caused by polarity, the transposon carried an outward-facing promoter designed to express downstream genes.  Mutants were selected on LB containing 60mg/mL tetracycline.  In our Tn-seq, the pool was found to contain approximately 110,000 unique insertion sites. More information can be found in PNAS. 2015 112(16):5189. doi: 10.1073/pnas.1422186112. Pools are sold in 1-mL aliquots with a titer of more than 10^9 cells/mL.  Pricing is shown on the relevant order form.

Ordering complete arrayed library copies and pooled libraries.

Complete arrayed library and pooled library orders may be initiated either by contacting us at pamutant [at] uw.edu or by filling out the Excel order form (instructions are on the first sheet) and emailing the form to pamutant [at] uw.edu.  Current pricing is shown on the relevant order form (academic or nonacademic).

In making replicates of the entire library for distribution, several steps of quality control are performed.  Strains are assessed for their growth by visual assessment of turbidity, and strains that did not grow well are grown up individually and included in supplemental plates.  Sanger sequencing is performed on a subset of wells (~3% of the library) to ensure plate orientation and library integrity (typically <2% of mutants provide unexpected sequence).

Mutant Names and Genotypes

In publications, please reference strains from the Two-Allele Library by strain name (PW####). 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 Held et al. 2012, J. Bacteriol. 194:6387 on publications resulting from the use of these strains.
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Academic institution order form
Non-academic institution order form
PA two-allele library (Excel spreadsheet)
Additional information about the PA Two allele Library