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High-throughput, quantitative analyses of genetic interactions in E. coli

Nature Methods volume 5, pages 781–787 (2008)Cite this article

Abstract

Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor–driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli.

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Figure 1: A flowchart depicting the different steps used in GIANT-coli.
Figure 2: A 12 × 12 genetic interaction matrix to validate GIANT-coli.
Figure 3: A toolkit that facilitates the use of GIANT-coli in genome-wide analyses.
Figure 4: Genome-wide screens using GIANT-coli.

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Acknowledgements

We thank S. Lee, A. Wong and J. Lee for technical assistance, and C.J. Ingles, P.J. Kiley, C. Squires, S.A. Johnson, A. Hochschild & T.J. Silhavy for critically reading this manuscript and offering useful suggestions. This work was supported by Sandler Family Funding (to C.A.G. and to N.J.K.), US National Institutes of Health (GM036278 to C.A.G. and GM62662 to B.L.W.), Japanese Ministry of Education, Culture, Sports, Science and Technology (Core Research for Evolutional Science and Technology) Grant-in-Aid for Scientific Research and Grant-in-Aid for Scientific Research on Priority Areas (to H.M.). A.T. is a recipient of a European Molecular Biology Organization fellowship.

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of California at San Francisco, 600 16th Street, San Francisco, 94158, California, USA

    Athanasios Typas, Robert J Nichols, Bentley Lim & Carol A Gross

  2. Department of Biology, Texas A&M University, 3258 TAMU College Station, 77843, Texas, USA

    Deborah A Siegele

  3. Department of Cellular and Molecular Pharmacology and The California Institute for Quantitative Biomedical Research, University of California at San Francisco, 1700 4th Street, San Francisco, 94158, California, USA

    Michael Shales, Sean R Collins, Hannes Braberg, Jonathan S Weissman & Nevan J Krogan

  4. Howard Hughes Medical Institute, University of California at San Francisco, 1700 4th Street, San Francisco, 94158, California, USA

    Sean R Collins & Jonathan S Weissman

  5. Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0101, Nara, Japan

    Natsuko Yamamoto, Rikiya Takeuchi & Hirotada Mori

  6. Department of Biological Sciences, Purdue University, LILY 1-228, West Lafayette, 47907, Indiana, USA

    Barry L Wanner

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  1. Athanasios Typas
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Contributions

A.T., R.J.N., D.A.S., J.S.W., N.J.K. and C.A.G. designed research; A.T., R.J.N., D.A.S. and B.L. executed research; A.T., R.J.N., M.S., S.C. and H.B. analyzed data; N.Y., R.T., B.L.W. and H.M. contributed new reagents (single-gene knockout libraries and CIP plasmids); A.T., N.J.K. and C.A.G. wrote the paper; R.J.N., M.S., B.L.W. and J.S.W. edited the paper.

Corresponding authors

Correspondence to Nevan J Krogan or Carol A Gross.

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Typas, A., Nichols, R., Siegele, D. et al. High-throughput, quantitative analyses of genetic interactions in E. coli. Nat Methods 5, 781–787 (2008). https://doi.org/10.1038/nmeth.1240

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