Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection

Tomoya Baba, Takeshi Ara, Miki Hasegawa, Yuki Takai, Yoshiko Okumura, Miki Baba, Kirill A. Datsenko, Masaru Tomita, Barry L. Wanner, Hirotada Mori

Research output: Contribution to journalArticlepeer-review

5641 Citations (Scopus)


We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants - the 'Keio collection' - provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (

Original languageEnglish
Article numbermsb4100050
Pages (from-to)2006.0008
JournalMolecular systems biology
Publication statusPublished - 2006 May 16


  • Bacterial functional genomics
  • E. coli/gene
  • Essential gene
  • Knockout mutants
  • Resources
  • Systems biology

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Agricultural and Biological Sciences(all)
  • Applied Mathematics


Dive into the research topics of 'Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection'. Together they form a unique fingerprint.

Cite this