Manoil Lab

Klebsiella pneumoniae Mutant Library

Obtaining mutants from the K. pneumoniae MKP103 transposon mutant library

The mutant library was made in a derivative of K. pneumoniae strain KPNIH1, an ST258 clinical isolate from a hospital outbreak (http://www.ncbi.nlm.nih.gov/bioproject/73191). The derivative (called MKP103) is deleted of the carbapenemase gene carried on a large conjugal plasmid (pKPQIL) in KPNIH1, and the plasmid is otherwise intact. The transposon used for mutagenesis is a derivative of Tn5 carrying a chloramphenicol resistance determinant (transposon T30), and mutants were generated using transposon-transposase complex (“transposome”) mutagenesis. The library has 2-3 separate insertions per gene, and strains have been colony purified and re-sequenced. The library has 12,000 mutants in it, corresponding to ~85% of the predicted KPNIH1 genes. Most of the unrepresented genes presumably correspond to genes essential for growth on the medium (LB + chloramphenicol) used to generate the mutants.

Strains from this 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. 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 (MKP103) and the wild-type (KPNIH1). A genome browser for KPNIH1 can be found at http://tools.uwgenomics.org/cgi-bin/pgat_klebsiella/elementlist.cgi?id=CP008827.

Individual strains 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 kpmutant [ a t ] u.washington.edu.  Please email your completed order form in Excel format to submit an order. Order forms submitted through any other method or format will not be processed. For requests from countries requiring import or other permits (e.g., Canada or Australia), the requestor must obtain the permits and email copies to kpmutant [ 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 kpmutant [ 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 >$400.

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 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 T30, use transposon-specific primer Pcm-140 (5’-CTGCGAAGTGATCTTCCGTCAC-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 kpmutant@u.washington.edu.

The table of strains carries information about each mutant in the three-allele library.  There is a second page to the Excel sheet that contains a legend for the headings. We apologize, but as a small research lab we cannot send out replacements if our transposon location has been mis-assigned.  The confirmation column contains information about our level of confidence in our assignment, and more information can be found in the notes column.

When ordering strains from the three-allele library, please include on the order form the “Item number”, “Strain Name/ Location” (e.g., “tnkp1_lr150110p03q180”), the “96 well location” and the “Locus” (e.g., “KPNIH1_08855”).  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.

Mutant naming:  In publications, please reference strains from the three-allele Library by Strain Name/ Location  (unique identifier) and refer to the genotype in the following way:  gene name (or Locus if there is no gene name)-well name (final three digits of the strain name) as the allele number::Transposon name (T30).  For example, for strain tnkp1_lr150110p03q105, the genotype is hisD105::T30, and for straintnkp1_lr150110p03q101, the genotype is KPNIH1_00420-101::T30.

 

Academic Institution order form
Non-Academic Institution order form
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