Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.
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 (http://ecoli.aist-nara.ac.jp/).
Baba
Tomoya
T
Institute for Advanced Biosciences, Keio University, Tsuruoka City, Yamagata, Japan.
Ara
Takeshi
T
Hasegawa
Miki
M
Takai
Yuki
Y
Okumura
Yoshiko
Y
Baba
Miki
M
Datsenko
Kirill A
KA
Tomita
Masaru
M
Wanner
Barry L
BL
Mori
Hirotada
H
D004926:Escherichia coli / Q000235:genetics*; D017353:Gene Deletion; D020407:Internet; D009154:Mutation; D030781:Organisms, Genetically Modified