基于蔗糖诱导的致死性在枯草芽孢杆菌中直接筛选克隆DNA。
Direct selection of cloned DNA in Bacillus subtilis based on sucrose-induced lethality.
作者信息
Bramucci M G, Nagarajan V
机构信息
Central Research and Development, Environmental Biotechnology, E. I. du Pont de Nemours, Inc., Wilmington, Delaware 19880-0328, USA.
出版信息
Appl Environ Microbiol. 1996 Nov;62(11):3948-53. doi: 10.1128/aem.62.11.3948-3953.1996.
Expression of the Bacillus subtilis or Bacillus amyloliquefaciens sacB gene in the presence of sucrose is lethal for a variety of bacteria. Sucrose-induced lethality can be used to select for inactivation of sacB by insertion of heterologous DNA in sensitive bacteria. This procedure has not been applicable to B. subtilis heretofore because expression of wild-type sacB is not detrimental to B. subtilis. The W29 mutation in the B. amyloliquefaciens sacB gene interferes with processing of the levansucrase signal peptide. The W29 mutation does not affect growth of B. subtilis in media lacking sucrose. However, this mutation inhibited growth of B. subtilis in media containing sucrose. Inactivation of the fructose polymerase activity encoded by sacB indicated that levan production was essential for sucrose-induced lethality. As a result, it was possible to select for cloned DNA in B. subtilis by insertional inactivation of the mutant sacB gene located on a multicopy plasmid vector in medium containing sucrose.
在蔗糖存在的情况下，枯草芽孢杆菌或解淀粉芽孢杆菌的 sacB 基因表达对多种细菌具有致死性。蔗糖诱导的致死性可用于通过在敏感细菌中插入异源 DNA 来选择 sacB 的失活。迄今为止，该方法不适用于枯草芽孢杆菌，因为野生型 sacB 的表达对枯草芽孢杆菌无害。解淀粉芽孢杆菌 sacB 基因中的 W29 突变会干扰果聚糖蔗糖酶信号肽的加工。W29 突变不影响枯草芽孢杆菌在缺乏蔗糖的培养基中的生长。然而，该突变抑制了枯草芽孢杆菌在含有蔗糖的培养基中的生长。对 sacB 编码的果糖聚合酶活性的失活表明，果聚糖的产生对于蔗糖诱导的致死性至关重要。因此，通过位于多拷贝质粒载体上的突变 sacB 基因在含有蔗糖的培养基中的插入失活，可以在枯草芽孢杆菌中选择克隆的 DNA。
Zotero 插件