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Updated: Jan 3, 2026
Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
Published on: January 8, 2015
[Optimizing Mycobacterium Recombineering System (pJV53) to Promote the Screening of the Mycobacterium Mutants]
Chuhan Wang1, Peng-Jiao Ma1, Tao Luo1
1Laboratory of Infection and Immunity, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, China.
Objective:
To establish a way for screening Mycobacterium mutants through adding the screening markers into pJV53.
Methods:
The sucrose counter selection gene SacB and mutant hygromycin-resistant gene hyg were inserted into pJV53; The recovery of the hygromycin-resistance indicated the successful homologous recombination in Mycobacterium smegmatis (Ms), which could serve as mutant screening marker; The sucrose counter selection could be used to screen the plasmid-free mutants.
Results:
The recombinant plasmid pJV53-SacB-hyg were successfully constructed. The rifampin-resistant rpoB D516Y and rpoB H526Q mutants and MSMEG_4487 G188A mutant were efficiently screened out. All mutants had shed the plasmid successfully.
Conclusion:
pJV53-SacB-hyg can efficiently contribute to construct and screen the mutants and to get the mutants shedding the plasmid self, which has high value of extensive application; the D516Y and H526Q mutations in gene rpoB of Mycobacterium tuberculosis contribute to its rifampin-resistance.
