A bacterial adenylate cyclase-based two-hybrid system compatible with gateway ® cloning

Macy G. Olson, Megan Goldammer, Emilie Gauliard, Daniel Ladant, Scot P. Ouellette

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

LanguageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages75-96
Number of pages22
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1794
ISSN (Print)1064-3745

Fingerprint

Adenylyl Cyclases
Organism Cloning
Adenylate Cyclase Toxin
Cyclic AMP
Proteins
Escherichia coli
Operon
Catalytic Domain
Membrane Proteins
Gene Expression

Keywords

  • Adenylate cyclase
  • Bacterial two-hybrid
  • BACTH
  • cAMP signaling
  • Gateway® cloning
  • Protein interaction assay

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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 Methods in Molecular Biology (pp. 75-96). (Methods in Molecular Biology; Vol. 1794). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7871-7_6

A bacterial adenylate cyclase-based two-hybrid system compatible with gateway ® cloning. / Olson, Macy G.; Goldammer, Megan; Gauliard, Emilie; Ladant, Daniel; Ouellette, Scot P.

Methods in Molecular Biology. Humana Press Inc., 2018. p. 75-96 (Methods in Molecular Biology; Vol. 1794).

Research output: Chapter in Book/Report/Conference proceedingChapter

Olson, MG, Goldammer, M, Gauliard, E, Ladant, D & Ouellette, SP 2018, A bacterial adenylate cyclase-based two-hybrid system compatible with gateway ® cloning. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1794, Humana Press Inc., pp. 75-96. https://doi.org/10.1007/978-1-4939-7871-7_6
Olson MG, Goldammer M, Gauliard E, Ladant D, Ouellette SP. A bacterial adenylate cyclase-based two-hybrid system compatible with gateway ® cloning. In Methods in Molecular Biology. Humana Press Inc. 2018. p. 75-96. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-7871-7_6
Olson, Macy G. ; Goldammer, Megan ; Gauliard, Emilie ; Ladant, Daniel ; Ouellette, Scot P. / A bacterial adenylate cyclase-based two-hybrid system compatible with gateway ® cloning. Methods in Molecular Biology. Humana Press Inc., 2018. pp. 75-96 (Methods in Molecular Biology).
@inbook{b9542a5a0a0f4d5e90404798eedad7c0,
title = "A bacterial adenylate cyclase-based two-hybrid system compatible with gateway {\circledR} cloning",
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{\circledR} recombinational cloning, BACTHGW. This chapter explains the principle of the BACTH, its Gateway{\circledR} modified system, and details of the general procedure.",
keywords = "Adenylate cyclase, Bacterial two-hybrid, BACTH, cAMP signaling, Gateway{\circledR} cloning, Protein interaction assay",
author = "Olson, {Macy G.} and Megan Goldammer and Emilie Gauliard and Daniel Ladant and Ouellette, {Scot P.}",
year = "2018",
month = "1",
day = "1",
doi = "10.1007/978-1-4939-7871-7_6",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "75--96",
booktitle = "Methods in Molecular Biology",

}

TY - CHAP

T1 - A bacterial adenylate cyclase-based two-hybrid system compatible with gateway ® cloning

AU - Olson, Macy G.

AU - Goldammer, Megan

AU - Gauliard, Emilie

AU - Ladant, Daniel

AU - Ouellette, Scot P.

PY - 2018/1/1

Y1 - 2018/1/1

N2 - 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.

AB - 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.

KW - Adenylate cyclase

KW - Bacterial two-hybrid

KW - BACTH

KW - cAMP signaling

KW - Gateway® cloning

KW - Protein interaction assay

UR - http://www.scopus.com/inward/record.url?scp=85048032854&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85048032854&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-7871-7_6

DO - 10.1007/978-1-4939-7871-7_6

M3 - Chapter

T3 - Methods in Molecular Biology

SP - 75

EP - 96

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -