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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >An efficient system for the generation of marked genetic mutants in members of the genus Burkholderia
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An efficient system for the generation of marked genetic mutants in members of the genus Burkholderia

机译：在伯克德列利亚属成员中产生明显的遗传突变体的有效系统

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To elucidate the function of a gene in bacteria it is vital that targeted gene inactivation (allelic replacement) can be achieved. Allelic replacement is often carried out by disruption of the gene of interest by insertion of an antibiotic-resistance marker followed by subsequent transfer of the mutant allele to the genome of the host organism in place of the wild-type gene. However, due to their intrinsic resistance to many antibiotics only selected antibiotic-resistance markers can be used in members of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc). Here we describe the construction of improved antibiotic-resistance cassettes that specify resistance to kanamycin, chloramphenicol or trimethoprim effectively in the Bcc and related species. These were then used in combination with and/or to construct a series enhanced suicide vectors, pSHAFT2, pSHAFT3 and pSHAFT-GFP to facilitate effective allelic replacement in the Bcc. Validation of these improved suicide vectors was demonstrated by the genetic inactivation of selected genes in the Bcc species Burkholderia cenocepacia and B. lata, and in the non-Bcc species, B. thailandensis. (C) 2016 The Authors. Published by Elsevier Inc.
机译：为了阐明细菌中基因的功能，可以实现靶向基因失活（等位基因替代）至关重要的是。等位基因替代通常通过插入抗生素抗性标记而受到利益基因的破坏，然后随后将突变等位基因转移到宿主生物的基因组代替野生型基因。然而，由于它们对许多抗生素的内在抗性，只有选择的抗生素抗性标记物可用于Burkowneria属的成员，包括BurkholderiaCepacia复合物（BCC）。在这里，我们描述了改进的抗生素抗性磁带的构建，其在BCC和相关物种中有效地指定对卡那霉素，氯霉素或三甲基丙杆菌的抗性。然后将这些与其组合使用和/或构建系列增强的自杀式载体，PShaft2，PShaft3和PShaft-GFP，以促进BCC中有效的等位基因替代。通过BCC物种Burkhowderia Cenocepacia和B. Lata的选定基因的遗传失活来证明了这些改进的自杀载体的验证，并在非BCC物种中，B.泰国人。 （c）2016年作者。 elsevier公司发布

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