Genetic Manipulation of Klebsiella pneumoniae.
Genetic Manipulation of Klebsiella pneumoniae.
复制标题
肺炎克雷伯菌的基因操作。
DOI：
10.1002/cpz1.912
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Mike,LauraA
中科院分区：
文献类型：
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作者：
Ring,BrookeE;Khadka,Saroj;Pariseau,DrewA;Mike,LauraA
肺炎克雷伯菌是一种革兰氏阴性的棒状细菌，常见于人类肠道。虽然它通常作为正常菌群的一部分存在，但它也可能引起与医疗保健相关的感染，并造成严重后果。通过基因操作了解导致其毒性的特定基因对于潜在的治疗干预至关重要。然而,manipulatingK。肺炎由于其胞外多糖胶囊而面临挑战。这篇文章提出了一个全面的协议集合，旨在促进基因操作的k。肺炎。通过遵循这些方案，研究人员将获得必要的技能来制备电致能细胞，利用电穿孔高效导入质粒DNA，使用λ Red重组酶系统构建等基因突变体，并生成用于恢复敲除菌株表型性状的互补载体。这些协议提供了有价值的工具和技术来导航与研究和修改k相关的复杂性。肺炎。©2023作者。Wiley期刊有限责任公司出版的现行协议。基本协议1：制备电胜任物。备选方案1：制备电竞液重组肺炎细胞基本方案2：转化基本方案3：构建等基因突变体支持方案1：通过集落pcr确认基因敲除支持方案2：验证继发性突变的缺失基本方案4：产生未标记的基因敲除突变体基本方案5：构建一个互补载体叉。肺炎
Klebsiella pneumoniaeis a Gram‐negative, rod‐shaped bacterium commonly found in the human intestine. Although it typically exists as part of the normal flora, it can also cause healthcare‐associated infections with severe consequences. Understanding the specific genes responsible for its virulence through genetic manipulation is crucial for potential therapeutic interventions. However, manipulatingK. pneumoniaepresents challenges due to its exopolysaccharide capsule. This article presents a comprehensive collection of protocols designed to facilitate the genetic manipulation ofK. pneumoniae. By following these protocols, researchers will acquire the necessary skills to prepare electrocompetent cells, utilize electroporation for efficient plasmid DNA introduction, construct isogenic mutants using the λ Red recombinase system, and generate a complementation vector for restoring the phenotypic traits of knockout strains. These protocols provide valuable tools and techniques to navigate the intricacies associated with studying and modifyingK. pneumoniae. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Preparing electrocompetentK. pneumoniaecellsAlternate Protocol 1: Preparing electrocompetentK. pneumoniaecells for recombineeringBasic Protocol 2: TransformingK. pneumoniaeusing electroporationBasic Protocol 3: Constructing isogenic mutants inK. pneumoniaeusing the λ Red recombinase systemSupport Protocol 1: Confirming a knockout via colony PCRSupport Protocol 2: Verifying absence of secondary mutationsBasic Protocol 4: Generating unmarked knockout mutants inK. pneumoniaeusing the pFLP plasmidBasic Protocol 5: Constructing a complementation vector forK. pneumoniae