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

Macy Olson (1, 2) , Megan Goldammer (1) , Emilie Gauliard (3) , Daniel Ladant (3) , Scot Ouellette (1, 2)

1 USD - University of South Dakota [Vermillion]
2 University of Nebraska Medical Center
3 Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions

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.

Keywords: Adenylate cyclase; BACTH; Bacterial two-hybrid; Gateway® cloning; Protein interaction assay; cAMP signaling

Domains: Life Sciences [q-bio]

Macy Olson, Megan Goldammer, Emilie Gauliard, Daniel Ladant, Scot Ouellette. A Bacterial Adenylate Cyclase-Based Two-Hybrid System Compatible with Gateway® Cloning. Springer. Methods in Molecular Biology, 1794, Humana Press, pp.75-96, 2018, Two-Hybrid Systems, 978-1-4939-7870-0. ⟨10.1007/978-1-4939-7871-7_6⟩. ⟨pasteur-02559575⟩