Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: the Keio collection
Open accessAbstract
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/).
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CyDisCo has been exploited as an efficient route for production of scFv and Fab fragments derived from known antibodies of different classes and organisms in the cytoplasm of the KEIO K12 E. coli strain in shake flasks.
“The CyDisCo has been exploited as an efficient route for the production of scFv and Fab fragments derived from known antibodies of different classes and from different organisms (human, mouse and humanized) in the cytoplasm of the KEIO (collection parental K12) E. coli strain in shake flasks [127].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [127]
The Results and discussion section (“Keio collection mutants”; page unavailable) identifies BW25113 as the E. coli K-12 strain used to construct the Keio collection, supporting the strain identification. It does not report CyDisCo production of scFv or Fab fragments, the antibody origins, or shake-flask production, so it does not support the sentence’s main production claim. (LLM, conf=0.86)
Experimental — accuracy not yet measuredSingle-molecule sensitivity of the imaging was confirmed by abrupt photobleaching of membrane-bound YFPs expressed at low level.
“Single molecule sensitivity was confirmed by abrupt photobleaching of membrane-bound YFPs expressed at low level (Fig. S14) (8, 18, 23).”
From Quantifying <i>E. coli</i> Proteome and Transcriptome with Single-Molecule Sensitivity in Single Cells (2010) · cites this paper as [23]
The Results and discussion passage on deletion-mutant verification describes PCR checks, while Materials and methods > Growth tests describes absorbance measurements; neither addresses single-molecule imaging. Searches for photobleaching, membrane-bound YFP, and fluorescence imaging found no passage confirming the claimed sensitivity test. Page numbers were not available in our copy. (LLM, conf=0.78)
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- Authors
- Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K. A., Tomita, M., Wanner, B. L., & Mori, H.
- Affiliations
- Institute for Advanced Biosciences, Keio University (JP)
- Japan Science and Technology Agency (JP)
- Purdue University West Lafayette (US)
- Corresponding author
- Wanner, Barry L
- Mori, Hirotada
- Venue
- Molecular Systems Biology
- Published
- 2006-02-21
- Volume
- 2
- Issue
- 1
- Pages
- 2006.0008-2006.0008
- ISSN
- 1744-4292 (ISSN-L)
- PMC
- PMC1681482
- OpenAlex
- W2111967267
- Cited by
- 7,858
- References
- 196
- Citations by year
- 2026: 331 · 2025: 452 · 2024: 489 · 2023: 429 · 2022: 458 · 2021: 490 · 2020: 502 · 2019: 451 · 2018: 454 · 2017: 464 · 2016: 394 · 2015: 435 · 2014: 451 · 2013: 415 · 2012: 376
- FWCI
- 117.12
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Molecular Systems Biology (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Keio University
- National Institutes of Health
- National Institute of General Medical Sciences (GM 62662, R01GM062662)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Keywords
- Keio collection, Escherichia coli K-12, single gene deletion, non-essential genes, kanamycin resistance cassette, gene inactivation, gene function analysis, gene regulatory networks, essential genes
- MeSH terms
- Escherichia coli, Mutation, Gene Deletion, Internet, Organisms, Genetically Modified
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