Abstract
Research on mycobacterial genetics relies heavily on techniques for directed gene mutation, but genetic studies are often hampered by the difficulty of generating gene deletions in mycobacteria. We developed an efficient and improved deletion system, described here in detail, which can be used to construct multiple unmarked recombinants in mycobacteria. We tested this system by using it to sequentially delete four pairs of toxin-antitoxin genes in Mycobacterium smegmatis.
Highlights
Research on mycobacterial genetics relies heavily on techniques for directed gene mutation, but genetic studies are often hampered by the difficulty of generating gene deletions in mycobacteria
1) Following recombination, cells cured of pJV53-GFP could be identified (Fig. 2); 2) The Zeo-resistance gene cassette provides a new option for targeted strains that already harbor a Hyg-resistance gene; 3) The Hyg- and Zeo-resistance cassettes can be used in alternation when multiple genes are to be deleted sequentially, by which it can save the time for curing antibiotic gene
An additional step is required to cure the antibiotic gene is needed before the gene can be deleted if only one antibiotic resistance cassette is used
Summary
Research on mycobacterial genetics relies heavily on techniques for directed gene mutation, but genetic studies are often hampered by the difficulty of generating gene deletions in mycobacteria. Genus Mycobacterium includes the pathogenic species tuberculosis and leprae, as well as many other opportunistic pathogens that occupy specific environmental niches Both scientific study and medical control of mycobacteria require efficient molecular tools for various types of genetic manipulation, especially directed mutagenesis and gene deletion[1]. A wide range of genetic manipulations using suicide plasmids, non-replicating plasmids, specialized transduction with mycobacteriophages, counter-selectable markers, and long linear DNA fragments have been developed to obtain mycobacterial mutants[2,3,4,5,6] These methods typically yield small numbers of mutants and high background levels of false-positive colonies[7]. We tested this system and demonstrated that it can be used for sequential deletion of multiple genes in M. smegmatis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
