Manoil Lab

Acinetobacter baumannii Mutant Library

The Acinetobacter baumannii cost center will resume distribution of individual mutants in mid-January, 2019, and will accept orders from the beginning of the year. Single mutant distribution will be permanently discontinued July 1, 2019. Unfortunately, we have had to increase the fee per strain; please use the current order form with the new pricing.

Obtaining mutants from the A. baumannii AB5075 transposon mutant library (Gallagher et al. 2015)


Strains from the AB5075 three-allele mutant library are available to the research community through a nonprofit cost center at the University of Washington. The accompanying table lists the mutants available.  You may also visit the Transposon Mutant Library Browser to facilitate browsing and selecting strains.  There are two to three mutants available for most nonessential genes.  The strains have been single colony-purified, and the insertion locations for most of them have been confirmed by re-sequencing.  Individual mutants are available, as is the parent strain (AB5075-UW).

Individual strains, transposon mutant pools, and ordered copies of the three-allele library are available for a charge.  To request individual mutants, fill out the order form that corresponds to your type of institution (links below) and send the filled-in Excel file to abmutant [ a t ] u.washington.edu.  For requests from countries requiring import or other permits (e.g., Canada or Australia), the requestor must obtain the permits and email copies to abmutant [ a t ] u.washington.edu. While email is preferred, if it is necessary to fax copies of permits, they may be faxed to Manoil lab at 206-685-7301.  To request the entire three-allele library, email abmutant [ a t ] u.washington.edu.

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 >$1000.

Maintaining strains.  Samples of the strains received should be maintained as frozen stocks (–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 after overnight growth scoop up a generous sample from the dense part of the plate for the frozen stock (in LB containing 5% DMSO).  Every strain is viable at the time of shipping. Once a strain has been shipped, there will be no refunds or reshipments without additional orders.

Reconfirming strains.  We 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 three-allele library, a small percentage of mutant insertion sites did not match the original assignments. We recommend that researchers test at least 5-10 isolated colonies by PCR for each strain for the appropriate insertion, because, while we have made every attempt to maintain purity of each mutant, high throughput growth and distribution may lead to some mixed cultures.  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 appropriate flanking primers.  These primers should be initially tested using the wild type parent strain.  To show that the intact gene is absent in the insertion strain, PCR with the same primers should yield either no band or a band corresponding to a much larger product.  To show the presence of the transposon insertion, use a transposon-specific primer with one of the flanking primers.  For transposon T26, use transposon-specific primer Pgro-172 (5’-TGAGCTTTTTAGCTCGACTAATCCAT-3'); for transposon T101, use transposon-specific primer hyg-174 (5’-GAAGCATTTATCAGGGTTATTGTCTCA-3’).  The flanking primer should be chosen according to the orientation of the transposon relative to the insertion site.  For ‘F’ insertions, the transposon-specific primer will point toward lower genome positions.  When you have either positively or negatively confirmed a mutant strain, please send an email reporting your result to abmutant [at] u.washington.edu.

The table of strains carries 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 Excel file.   We apologize, but as a small research lab we cannot send out replacements if our transposon location has been mis-assigned. 

Phil Rather's group has described a phase variation in A. baumannii AB5075 that affects colony morphology and other phenotypes (Tipton et al, J. Bacteriol. 197, 2593-2599 (2015)). Mutants from our three-allele library exhibit the variation, and it is important where feasible to employ strains of the same colony type when comparing phenotypes. Conditions for distinguishing colony types are described in the Tipton et al. publication.

When ordering strains from the three-allele library, please include on the order form the “Item #” (e.g., “AB00001”), the “Strain Name” (e.g., “tnab1_kr121213p03q180”), the “96 well location” (e.g., H10), and the “Ab Locus” (e.g., “ABUW_0001”).  Note that the shopping cart feature of the Transposon Mutant Library Browser automatically generates some of this information in list form for easy copying and pasting into the order form.

Ordered library copies: 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 naming: In publications, please reference strains from the Three-allele Library by Strain Name (unique identifier) and refer to the genotype in the following way:  gene name (or ABUW Locus if there is no gene name)-well name (final three digits of the location field) as the allele number::Transposon name (T26 or T101).  For example, for strain AB00024, the genotype is adeT127::T26, and for strain AB00033, the genotype is ABUW_0020-148::T101.

 

 

Please follow these links for additional information:

(If forms do not download using your browser, please try Chrome, Internet Explorer or Firefox.)


Academic institution order form
Non-academic institution order form
Ab three-allele library
Transposon Mutant Library Browser