title: Engineering more stable, selectable marker-free autoluminescent mycobacteria by one step.
abstract: In our previous study, we demonstrated that the use of the autoluminescent Mycobacterium tuberculosis as a reporter strain had the potential to drastically reduce the time, effort, animals and costs consumed in evaluation of the activities of drugs and vaccines in live mice. However, the strains were relatively unstable and lost reporter with time without selection. The kanamycin selection marker used wasn't the best choice as it provides resistance to amino glycosides which are an important class of second line drugs used in tuberculosis treatment. In addition, the marker could limit utility of the strains for screening of new potential drugs or evaluating drug combinations for tuberculosis treatment. Limited selection marker genes for mycobacterial genetic manipulation is a major drawback for such a marker-containing strain in many research fields. Therefore, selectable marker-free, more stable autoluminescent mycobacteria are highly needed. After trying several strategies, we created such mycobacterial strains successfully by using an integrative vector and removing both the resistance maker and integrase genes by Xer site-specific recombination in one step. The corresponding plasmid vectors developed in this study could be very convenient in constructing other selectable marker-free, more stable reporter mycobacteria with diverse applications.
authors.affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China.
authors.forename: Moses M
authors.initials: MM
authors.lastname: Njire
authors.affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China.
authors.forename: Tian
authors.initials: T
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China.
authors.forename: Bangxing
authors.initials: B
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China.
authors.forename: Tianzhou
authors.initials: T
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Cao
authors.affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China.
authors.forename: Junting
authors.initials: J
authors.lastname: Wan
authors.affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China.
authors.forename: Zhengchao
authors.initials: Z
authors.lastname: Tu
authors.affiliation: State Key Laboratory of Respiratory Disease, Department of Clinical Laboratory, The Guangzhou Chest Hospital, Guangzhou, Guangdong, China.
authors.forename: Yaoju
authors.initials: Y
authors.lastname: Tan
authors.affiliation: State Key Laboratory of Respiratory Disease, Department of Clinical Laboratory, The Guangzhou Chest Hospital, Guangzhou, Guangdong, China.
authors.forename: Shouyong
authors.initials: S
authors.lastname: Tan
authors.affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China.
authors.forename: Tianyu
authors.initials: T
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D055808:Drug Discovery / Q000191:economics; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D019426:Integrases / Q000235:genetics; D007613:Kanamycin Resistance; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D019426:Integrases