E. coli Keio Knockout Parent Strain - BW25113

The E. coli Keio Knockout Collection was designed to provide researchers with in-frame, single-gene deletion mutants for all non-essential E. coli K-12 genes.

The E. coli Keio Knockout Collection contains a set of single-gene, knockout mutants for all nonessential genes in E. coli K-12.

Developed as a collaboration between the Institute for Advanced Biosciences, Keio University (Japan), Nara Institute of Science and

Technology (Japan), and Purdue University, this collection of mutant strains was designed to create in-frame deletions upon excision of a kanamycin resistance cassette.

The E. coli Keio collection is not only a resource for systematic functional genomics, but can also serve as a tool for a number of reverse genetics approaches. In contrast to forward genetic approaches in which mutant phenotypes are associated with a corresponding gene, the Keio collection permits analysis of the consequences associated with the complete loss of gene function.

Highlights

• There are two independent deletion strains for each of 3985 genes, yielding a total of 7970 strains
• Each deleted gene is replaced with a kanamycin resistance cassette that can be excised by FLP recognition target (FRT) recombination
• Strain background: E. coli K-12 BW25113

Primer design and construction of single-gene deletion mutants in the Keio collection

Gene knockout primers have 20- to 30-nucleotide ends for priming upstream (P1) and downstream (P2) of the FRT sites flanking the kanamycin resistance gene in pKD13 and 50-nucleotide ends homologous to upstream (H1) and downstream (H2) chromosomal sequences for targeting the gene deletion. H1 includes the gene B (target) initiation codon. H2 includes codons for the six C-terminal residues, the stop codon, and 29 nucleotides downstream. The same primer design with respect to gene B was used to target deletions regardless of whether gene B lies in an operon with genes A and C, as shown, or in different chromosomal arrangements. Novel junctions created between the resistance cassette and neighboring upstream (gene A) and downstream (gene C) sequences were verified by PCR with kanamycin (k1 or k2) and locus-specific (U or D) primers. Structures created after excision of the resistance gene are verified by PCR with neighboring gene-specific primers and by direct DNA sequencing of the region encompassing the H1-P1-FRT-P2-H2 scar to verify correct ones, as described elsewhere (Datsenko and Wanner, 2000). SD, Shine–Dalgarno ribosome binding sequence. (Baba et al., 2004).

Mutagenesis of E. coli K-12 in the Keio collection

Of 4288 genes targeted, deletions were obtained for 3985 ORFs. Based on finding mutants with the predicted structure, these 3985 genes are likely nonessential, while the 303 genes (including 37 genes of unknown function), for which no mutants were found, are candidates for essential genes. (Baba et al., 2004)

Note

We provide certain clone resources developed by leading academic laboratories. Many of these resources address the needs of specialized research communities not served by other commercial entities. In order to provide these as a public resource, we depend on the contributing academic laboratories for quality control.

Therefore, these are distributed in the format provided by the contributing institution "as is" with no additional product validation or guarantee. We are not responsible for any errors or performance issues. Additional information can be found in the product manual as well as in associated published articles (if available). Alternatively, the source academic institution can be contacted directly for troubleshooting.

Entire Collection

The E. coli Keio Knockout Collection is provided in 96-well microtiter plates. These will ship on dry ice via overnight delivery and should be stored at –80°C immediately upon receipt.

Individual Clones

Clones are provided as a live culture in a 2 mL tube. Each tube contains LB medium supplemented with 8% glycerol and kanamycin. Within 2 to 3 business days of receiving your order we will ship your clone at room temperature via express delivery. Store the stock clone at –80°C.

References

1.T. Baba, et al, Construction of Escherichia coli K12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology. 2, 2006.0008 (21 February 2006). [doi:10.1038/msb4100050]
2.K.A. Datsenko, B.L. Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. PNAS. 97, 6640-6645 (2000).