首页    期刊浏览 2024年12月14日 星期六
登录注册

文章基本信息

  • 标题:A uniform cloning platform for mycobacterial genetics and protein production
  • 本地全文:下载
  • 作者:Fabian M. Arnold ; Michael Hohl ; Sille Remm
  • 期刊名称:Scientific Reports
  • 电子版ISSN:2045-2322
  • 出版年度:2018
  • 卷号:8
  • 期号:1
  • 页码:9539
  • DOI:10.1038/s41598-018-27687-5
  • 语种:English
  • 出版社:Springer Nature
  • 摘要:Molecular research on mycobacteria relies on a multitude of tools for the genetic manipulation of these clinically important bacteria. However, a uniform set of vectors allowing for standardized cloning procedures is not available. Here, we developed a versatile series of mycobacterial vectors for gene deletion, complementation and protein production and purification. The vectors are compatible with fragment exchange (FX) cloning, a recently developed high-throughput cloning principle taking advantage of the type IIS restriction enzyme SapI and its capacity to generate sticky trinucleotide ends outside of its recognition sequence. FX cloning allows for the efficient cloning into an entry vector and the facile transfer of the sequenced insert into a variety of destination vectors. We generated a set of mycobacterial expression vectors spanning a wide range of expression strengths, tagging variants and selection markers to rapidly screen for the optimal expression construct in order to purify membrane proteins from the model organism Mycobacterium smegmatis. Further, we generated a series of suicide vectors containing two counterselection markers and used them to delete twenty genes encoding for potential drug efflux pumps in M. smegmatis. The vectors will further facilitate genetic and biochemical research on various mycobacterial species.
国家哲学社会科学文献中心版权所有