A single step three-strain in vivo Gateway reaction.
B2H
Bacterial two-hybrid
CcdB
Cloning
DB3.1
E. coli
Gateway
In vivo
MFDpir
Plasmids
Transformation
Journal
Plasmid
ISSN: 1095-9890
Titre abrégé: Plasmid
Pays: United States
ID NLM: 7802221
Informations de publication
Date de publication:
11 2021
11 2021
Historique:
received:
21
08
2021
revised:
09
11
2021
accepted:
15
11
2021
pubmed:
22
11
2021
medline:
1
2
2022
entrez:
21
11
2021
Statut:
ppublish
Résumé
We developed a simplified, highly efficient Gateway reaction that recombines target DNA to expression (destination) plasmids in vivo and subsequently conjugates the final vector into a recipient strain, all in a single step. This recipient strain does not need to contain any selective marker and can be freely chosen as long as it is sensitive to ccdB counterselection and can be targeted by the RP4α conjugation system. Our protocol is simple, robust, and cost effective. It works in 96-well plate format and performs across a range of temperatures. We designed modular, minimal destination vectors containing a modified Gateway insert to ease vector design by providing locations for insertion of tags, promoters, or conjugations. To demonstrate the utility of our system, we created destination vectors with split adenylate cyclase tags for bacterial two-hybrid (B2H) studies and screened a library of diguanylate cyclases for protein-protein interactions in a single step.
Identifiants
pubmed: 34801582
pii: S0147-619X(21)00055-X
doi: 10.1016/j.plasmid.2021.102608
pii:
doi:
Substances chimiques
DNA
9007-49-2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
102608Informations de copyright
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.