Identification of Essential Genes and Synthetic Lethal Gene Combinations in Escherichia coli K-12

  • Hirotada MoriEmail author
  • Tomoya Baba
  • Katsushi Yokoyama
  • Rikiya Takeuchi
  • Wataru Nomura
  • Kazuichi Makishi
  • Yuta Otsuka
  • Hitomi Dose
  • Barry L. WannerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1279)


Here we describe the systematic identification of single genes and gene pairs, whose knockout causes lethality in Escherichia coli K-12. During construction of precise single-gene knockout library of E. coli K-12, we identified 328 essential gene candidates for growth in complex (LB) medium. Upon establishment of the Keio single-gene deletion library, we undertook the development of the ASKA single-gene deletion library carrying a different antibiotic resistance. In addition, we developed tools for identification of synthetic lethal gene combinations by systematic construction of double-gene knockout mutants. We introduce these methods herein.

Key words

Gene deletion Escherichia coli FLP recombinase FRT Gene knockout Homologous recombination In-frame deletion Lambda Red recombinase Essential genes KEIO collection ASKA collection 



This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and a grant from CREST, JST (Japan Science and Technology), NEDO (New Energy and Industrial Technology Development Organization), Tsuruoka City, Yamagata prefecture in Japan. B.L.W. is supported by the US National Science Foundation (grant number 106394).


  1. 1.
    Baba T, Ara T, Hasegawa M et al (2006) Construction of Escherichia coli K-12 in-frame, single-gene knock-out mutants—the Keio collection. Mol Syst Biol. doi: 10.1038/msb4100050 Google Scholar
  2. 2.
    Yamamoto N, Nakahigashi K, Nakamichi T et al (2009) Update on the Keio collection of Escherichia coli single-gene deletion mutants. Mol Syst Biol 5:335PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Giaever G, Chu AM, Ni L et al (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387–391PubMedCrossRefGoogle Scholar
  4. 4.
    Kobayashi K, Ehrlich SD, Albertini A et al (2003) Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A 100:4678–4683PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Jacobs MA, Alwood A, Thaipisuttikul I et al (2003) Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 100:14339–14344PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Mori H, Isono K, Horiuchi T et al (2000) Functional genomics of Escherichia coli in Japan. Res Microbiol 151:121–128PubMedCrossRefGoogle Scholar
  7. 7.
    Kohara Y, Akiyama K, Isono K (1987) The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell 50:495–508PubMedCrossRefGoogle Scholar
  8. 8.
    Miki T, Yamamoto Y, Matsuda H (2008) A novel, simple, high-throughput method for isolation of genome-wide transposon insertion mutants of Escherichia coli K-12. Methods Mol Biol 416:195–204PubMedCrossRefGoogle Scholar
  9. 9.
    Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Baba T, Mori H (2008) The construction of systematic in-frame, single-gene knockout mutant collection in Escherichia coli K-12. Methods Mol Biol 416:171–181PubMedCrossRefGoogle Scholar
  11. 11.
    Butland G, Babu M, Diaz-Mejia JJ et al (2008) eSGA: E. coli synthetic genetic array analysis. Nat Methods 5:789–795PubMedCrossRefGoogle Scholar
  12. 12.
    Typas A, Nichols RJ, Siegele DA et al (2008) High-throughput, quantitative analyses of genetic interactions in E. coli. Nat Methods 5:781–787PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Gerdes SY, Scholle MD, Campbell JW et al (2003) Experimental determination and system level analysis of essential genes in Escherichia coli MG1655. J Bacteriol 185:5673–5684PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    van Opijnen T, Camilli A (2013) Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol 11:435–442PubMedCrossRefGoogle Scholar
  15. 15.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  16. 16.
    Takeuchi R, Tamura T, Nakayashiki T et al (2014) Colony-live—a high-throughput method for measuring microbial colony growth kinetics—reveals diverse growth effects of gene knockouts in Escherichia coli. BMC Microbiol 14:171PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Yong HT, Yamamoto N, Takeuchi R et al (2013) Development of a system for discovery of genetic interactions for essential genes in Escherichia coli K-12. Genes Genet Syst 88:233–240PubMedGoogle Scholar
  18. 18.
    Hiraga S (1986) Mechanisms of stable plasmid inheritance. Adv Biophys 21:91–103PubMedCrossRefGoogle Scholar
  19. 19.
    Kline BC (1985) A review of mini-F plasmid maintenance. Plasmid 14:1–16PubMedCrossRefGoogle Scholar
  20. 20.
    Hiraga S, Ogura T, Mori H et al (1985) Mechanisms essential for stable inheritance of mini-F plasmid. Basic Life Sci 30:469–487PubMedGoogle Scholar
  21. 21.
    Timmis K, Cabello F, Cohen SN (1975) Cloning, isolation, and characterization of replication regions of complex plasmid genomes. Proc Natl Acad Sci U S A 72:2242–2246PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Lovett MA, Helinski DR (1976) Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F’kn plasmid. J Bacteriol 127:982–987PubMedCentralPubMedGoogle Scholar
  23. 23.
    Lane HE (1981) Replication and incompatibility of F and plasmids in the IncFI Group. Plasmid 5:100–126PubMedCrossRefGoogle Scholar
  24. 24.
    Mori H, Kondo A, Ohshima A et al (1986) Structure and function of the F plasmid genes essential for partitioning. J Mol Biol 192:1–15PubMedCrossRefGoogle Scholar
  25. 25.
    Kitagawa M, Ara T, Arifuzzaman M et al (2005) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 12:291–299PubMedCrossRefGoogle Scholar
  26. 26.
    Rajagopala SV, Yamamoto N, Zweifel AE et al (2010) The Escherichia coli K-12 ORFeome: a resource for comparative molecular microbiology. BMC Genomics 11:470PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Nichols RJ, Sen S, Choo YJ et al (2011) Phenotypic landscape of a bacterial cell. Cell 144:143–156PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Tohsato Y, Mori H (2008) Phenotype profiling of single gene deletion mutants of E. coli using Biolog technology. Genome Inform 21:42–52PubMedCrossRefGoogle Scholar
  29. 29.
    Hayashi K, Morooka N, Yamamoto Y et al (2006) Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110. Mol Syst Biol. doi: 10.1038/msb4100049 PubMedCentralPubMedGoogle Scholar
  30. 30.
    Riley M, Abe T, Arnaud MB et al (2006) Escherichia coli K-12: a cooperatively developed annotation snapshot—2005. Nucleic Acids Res 34:1–9PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Murakami A, Nakatogawa H, Ito K (2004) Translation arrest of SecM is essential for the basal and regulated expression of SecA. Proc Natl Acad Sci U S A 101:12330–12335PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Nakatogawa H, Murakami A, Mori H et al (2005) SecM facilitates translocase function of SecA by localizing its biosynthesis. Genes Dev 19:436–444PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Kruse T, Moller-Jensen J, Lobner-Olesen A et al (2003) Dysfunctional MreB inhibits chromosome segregation in Escherichia coli. EMBO J 22:5283–5292PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Kruse T, Bork-Jensen J, Gerdes K (2005) The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex. Mol Microbiol 55:78–89PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Hirotada Mori
    • 1
    Email author
  • Tomoya Baba
    • 2
  • Katsushi Yokoyama
    • 1
  • Rikiya Takeuchi
    • 1
  • Wataru Nomura
    • 1
  • Kazuichi Makishi
    • 1
  • Yuta Otsuka
    • 1
  • Hitomi Dose
    • 1
  • Barry L. Wanner
    • 3
    Email author
  1. 1.Graduate School of Biological SciencesNara Institute of Science and TechnologyIkomaJapan
  2. 2.Transdiscplinary Research Integration CenterMishimaJapan
  3. 3.Department of Microbiology and ImmunobiologyHarvard Medical SchoolBostonUSA

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