New cloning vectors to facilitate quick allelic exchange in gram-negative bacteria

• Published in: BioTechniques Vol. 70; no. 2; pp. 116 - 119
• Main Authors: Mejia-Santana, Adrian, Lloyd, Cameron J, Klose, Karl E
• Format: Journal Article
• Language: English
• Published: England Future Science Ltd 01.02.2021
• Subjects: cloning; genetic engineering; gram-negative; microbiology; microbiology; gram-negative; cloning; genetic engineering
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/btn-2020-0135

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 gene provides counterselection for allelic exchange. The vectors have incorporated the alpha fragment with an enhanced multicloning site for easy blue/white screening and priming sites identified for efficient  assembly or other DNA assembly cloning techniques. Different antibiotic resistance markers allow versatility for use with different bacteria, and transformation into an 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 and . New plasmid vectors were designed to streamline allelic exchange in gram-negative bacteria by allowing for blue/white screening and restriction digest-independent cloning techniques as well as different antibiotic resistance genes to expand their utility to different bacteria.