Hansen C R, Khatiwara A, Ziprin R, Kwon Y M
Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, USA.
Lett Appl Microbiol. 2007 Dec;45(6):599-603. doi: 10.1111/j.1472-765X.2007.02232.x. Epub 2007 Oct 1.
To develop a novel method for rapid construction of Campylobacter jejuni deletion mutants.
We used overlapping extension PCR protocol to amplify a target sequence region of Camp. jejuni genomic DNA in which an internal fragment, Cj0618 coding sequence, was replaced by a chloramphenicol resistance cassette. After the resulting PCR product was introduced into electrocompetent Camp. jejuni 81-176, chloramphenicol-resistant mutants in which the wild type allele has been replaced by the deletion cassette were selected. DNA sequencing confirmed precise deletion in the Cj0618 gene. As expected from the previously reported role of Cj0618 in chick colonization, the resulting deletion mutant showed a caecal colonization defect in chick infection.
This method can be used for rapid construction of Camp. jejuni deletion mutants.
The use of this method should facilitate functional characterization of various Camp. jejuni genes.
开发一种快速构建空肠弯曲菌缺失突变体的新方法。
我们使用重叠延伸PCR方案扩增空肠弯曲菌基因组DNA的目标序列区域，其中内部片段Cj0618编码序列被氯霉素抗性盒取代。将所得PCR产物导入电转化的空肠弯曲菌81 - 176后，选择野生型等位基因已被缺失盒取代的氯霉素抗性突变体。DNA测序证实Cj0618基因发生了精确缺失。正如之前报道的Cj0618在鸡定殖中的作用所预期的那样，所得缺失突变体在鸡感染中表现出盲肠定殖缺陷。
该方法可用于快速构建空肠弯曲菌缺失突变体。
该方法的使用应有助于对空肠弯曲菌各种基因进行功能表征。
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