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

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

Abstract

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 

Notes

Acknowledgements

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).

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Hirotada Mori
    • 1
  • 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
  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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