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A Bacterial Adenylate Cyclase-Based Two-Hybrid System Compatible with Gateway® Cloning

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Two-Hybrid Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1794))

Abstract

The bacterial adenylate cyclase two-hybrid system (BACTH) is a genetic approach used to test protein interactions in vivo in E. coli. This system takes advantage of the two catalytic domains of Bordetella pertussis adenylate cyclase (CyaA) toxin, which can be fused separately to proteins of interest. If the proteins of interest interact, then the adenylate cyclase domains will be brought in close proximity to each other, reconstituting cyclic AMP (cAMP) production. Interacting proteins can be both qualitatively and quantitatively assessed by the expression of chromosomal genes of the E. coli lac or mal operon, which are positively regulated by cAMP production. Because cAMP is diffusible, the proteins of interest do not need to interact near the transcriptional machinery. Consequently, both cytosolic and membrane protein–protein interactions can be tested. The BACTH system has recently been modified to be compatible with Gateway® recombinational cloning, BACTHGW. This chapter explains the principle of the BACTH, its Gateway® modified system, and details of the general procedure.

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Acknowledgments

This work was supported in part by the University of South Dakota, Sanford School of Medicine, Division of Basic Biomedical Sciences, the University of Nebraska Medical Center, and by the Institut Pasteur and the Centre National de la Recherche Scientifique (CNRS UMR 3528, Biologie Structurale et Agents Infectieux). S.P.O. is supported by an award (1R35GM124798-01) from NIGMS/NIH.

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Authors and Affiliations

  1. Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD, USA

    Macy G. Olson, Megan Goldammer & Scot P. Ouellette

  2. Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, NE, USA

    Macy G. Olson & Scot P. Ouellette

  3. Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, Institut Pasteur, CNRS, UMR 3528, Paris, France

    Emilie Gauliard & Daniel Ladant

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  1. Macy G. Olson
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  2. Megan Goldammer
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  3. Emilie Gauliard
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  4. Daniel Ladant
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  5. Scot P. Ouellette
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Corresponding author

Correspondence to Scot P. Ouellette .

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Editors and Affiliations

  1. Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM)–Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, Madrid, Spain

    Luis Oñate-Sánchez

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Olson, M.G., Goldammer, M., Gauliard, E., Ladant, D., Ouellette, S.P. (2018). A Bacterial Adenylate Cyclase-Based Two-Hybrid System Compatible with Gateway® Cloning. In: Oñate-Sánchez, L. (eds) Two-Hybrid Systems. Methods in Molecular Biology, vol 1794. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7871-7_6

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  • DOI: https://doi.org/10.1007/978-1-4939-7871-7_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7870-0

  • Online ISBN: 978-1-4939-7871-7

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