Manoil Lab

Burkholderia thailandensis Mutant Library

The B. thailandensis 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 Burkholderia thailandensis E264 transposon mutant library (Gallagher et al. 2013, mBio 4:6)

Strains from the Seattle B. thailandensis E264 transposon 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. The strains have been single colony-purified, and there are two mutants available for most nonessential genes. Individual mutants are available. 

Individual strains are available for a charge.   To request a mutant, fill out the order form that corresponds to your type of institution (links below) and send the filled-in Excel file to btmutant [ 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 btmutant [ a t ] u.washington.edu (preferred) or fax copies to Manoil lab 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 >$1000

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 after overnight growth scoop up a generous sample from the dense part of the plate for the frozen stock (in LB containing 10% glycerol (w/v)). 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. 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 two-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 probably leads 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, which should be tested initially using the wild type parent strain. 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. For both T8 (ISlacZ hah-Tc) , and T23 (ISlacZ-PrhaBo-Tp/FRT) transposons, 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 insertion site. When you have either positively or negatively confirmed a mutant strain, please send an email to btmutant [ a t ] u.washington.edu conveying the results. 

The table of strains carries information about each mutant in the two-allele library. The second sheet of the Excel file has a description of each column heading.  We apologize, but as a small research lab we cannot send out replacements if our transposon location has been mis-assigned. 

When ordering strains from the BT Two-Allele Library, please include the “Item number”, “Strain name/location”, “96 well location”, and the “BT ORF” (e.g., “BTH_I0155”) from the “BT two Allele Library” Excel sheet on your 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).

In publications, please reference strains from the Two-Allele Library by strain name (unique identifier) and refer to the genotype in the following way: gene name (or BTORF if there is no gene name)-well name (final three digits of the location field) as the allele number::Transposon name (ISlacZ/hah-Tc or ISlacZ-PrhaBo-Tp/FRT).  For example, [for strain BT00042, the genotype is  hslU142:: ISlacZ/hah-Tc, and for strain BT00001, the genotype is BTH_I0155-101::ISlacZ/hah-Tc.]

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Academic institution order form
Non-academic institution order form
Bt two-allele library
Transposon Mutant Library Browser