Open AccessNew cloning vectors to facilitate quick allelic exchange in gram-negative bacteria
Author(s) -
Adrian Mejia-Santana,
Cameron J. Lloyd,
Karl E. Klose
Publication year - 2021
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/btn-2020-0135
Subject(s) - biology , transformation (genetics) , cloning (programming) , plasmid , vibrio cholerae , genetics , bacteria , escherichia coli , multiple cloning site , cloning vector , vibrio , gene , molecular cloning , microbiology and biotechnology , computational biology , vector (molecular biology) , recombinant dna , complementary dna , computer science , programming language
New cloning vectors have been developed with features to enhance quick allelic exchange in gram-negative bacteria. The conditionally replicative R6K and transfer origins facilitate conjugation and chromosomal integration into a variety of bacterial species, whereas the sacB gene provides counterselection for allelic exchange. The vectors have incorporated the lacZ alpha fragment with an enhanced multicloning site for easy blue/white screening and priming sites identified for efficient in vivo assembly or other DNA assembly cloning techniques. Different antibiotic resistance markers allow versatility for use with different bacteria, and transformation into an Escherichia coli strain capable of conjugation enables a quick method for allelic exchange. As a proof of principle, the authors used these vectors to inactivate genes in Vibrio cholerae and Salmonella typhimurium.