Improved Method for Transformation of Vibrio vulnificus by Electroporation.

Jane M Jayakumar, Orr H Shapiro, Salvador Almagro-Moreno
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引用次数: 2

Abstract

Vibrio vulnificus, an emergent human pathogen, causes fulminant septicemia with a mortality rate of over 50%. Unlike for other pathogenic Vibrio species, the factors to conclusively indicate the virulence potential of V. vulnificus strains remain largely unknown. Understanding the pathogenesis of this bacterium at a molecular level is severely hindered by inefficiencies in transformation, for instance, due to the presence of a periplasmic nuclease, Vvn. Currently, successful transformation of V. vulnificus is nearly impossible due to lack of mobilizable plasmids for the bacterium, requiring (i) very high DNA concentrations, (ii) plasmid linearization, (iii) development of novel V. vulnificus-derived plasmids, or (iv) time-consuming conjugation-based methods. To overcome these limitations, we describe a rapid, efficient, and reproducible electroporation protocol to effectively transform widely available plasmids, with different copy numbers and antibiotic resistances, into phylogenetically distant strains of V. vulnificus. Cells are made competent in high concentrations of sucrose devoid of cations and recovered from electroporation using a high-salinity recovery medium. Compared to existing methods for transformation of V. vulnificus, significantly higher efficiencies are obtained using this improved protocol. Rapid and effective transformations can markedly improve molecular analyses of V. vulnificus leading to a greater understanding of its virulence potential. This is crucial to develop rapid detection methods which have the potential to prevent future outbreaks. The electroporation protocol described here may be particularly useful for optimizing transformation of other nuclease-producing bacteria. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent cells Basic Protocol 2: Transformation of cells by electroporation.

电穿孔法转化创伤弧菌的改进方法。
创伤弧菌是一种紧急的人类病原体,可引起暴发性败血症,死亡率超过50%。与其他致病性弧菌不同,最终表明创伤弧菌菌株毒力潜力的因素在很大程度上仍然未知。在分子水平上理解这种细菌的发病机制受到转化效率低下的严重阻碍,例如,由于存在质周核酸酶,Vvn。目前,由于缺乏可动员的质粒,创伤弧菌的成功转化几乎是不可能的,这需要(i)非常高的DNA浓度,(ii)质粒线性化,(iii)开发新的创伤弧菌衍生的质粒,或者(iv)耗时的基于偶联的方法。为了克服这些限制,我们描述了一种快速、高效、可重复的电穿孔方案,以有效地将广泛可用的具有不同拷贝数和抗生素耐药性的质粒转化为系统发育上遥远的创伤弧菌菌株。细胞在缺乏阳离子的高浓度蔗糖中被制成能态,并使用高盐度的回收介质从电穿孔中回收。与现有的创伤弧菌转化方法相比,使用改进的方案获得了显着更高的效率。快速有效的转化可以显著改善创伤弧菌的分子分析,从而更好地了解其毒力潜力。这对于开发有可能预防未来疫情的快速检测方法至关重要。这里描述的电穿孔方案可能对优化其他核酸酶产生细菌的转化特别有用。©2020 Wiley期刊有限责任公司基本方案1:制备能态细胞基本方案2:通过电穿孔细胞转化。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Current Protocols in Microbiology
Current Protocols in Microbiology Immunology and Microbiology-Parasitology
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期刊介绍: Current Protocols in Microbiology provides detailed, step-by-step instructions for analyzing bacteria, animal and plant viruses, fungi, protozoans and other microbes. It offers updated coverage of emerging technologies and concepts, such as biofilms, quorum sensing and quantitative PCR, as well as proteomic and genomic methods. It is the first comprehensive source of high-quality microbiology protocols that reflects and incorporates the new mandates and capabilities of this robust and rapidly evolving discipline.
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