Microbial Gene Essentiality: Protocols and Bioinformatics

Volume 416 of the series Methods in Molecular Biology™ pp 171-181

The Construction of Systematic In-Frame, Single-Gene Knockout Mutant Collection in Escherichia coli K-12

  • Tomoya BabaAffiliated withNara Institute of Science and Technology (NAIST), Graduate School of Biological Sciences
  • , Hirotada MoriAffiliated withNara Institute of Science and TechnologyInstitute of Advanced Biosciences, Keio University

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Here we describe the systematic construction of well-defined, in-frame, single-gene deletions of all nonessential genes in Escherichia coli K-12. The principal strategy is based on the method for one-step inactivation of chromosomal genes in E. coli K-12 established by Datsenko and Wanner (1), namely, the replacement of a target gene with a selectable antibiotic-resistant marker generated by polymerase chain reaction (PCR) using oligonucleotide DNA primers homologous to the gene flanking regions. The advantages of this method include complete deletion of an entire open reading frame and precise design eliminating polar effects for the downstream genes on E. coli chromosome.

Key Words

complete deletion Escherichia coli FLP recombinase FRT gene knockout homologous recombination in-frame deletion lambda Red recombinase mutant site-specific recombination