Genetic Manipulation of Vibrio fischeri

Current Protocols in Microbiology Pub Date : 2020-09-25 DOI:10.1002/cpmc.115

David G. Christensen, Jovanka Tepavčević, Karen L. Visick

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引用次数: 7

Abstract

Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species. The bacterium has been used as a model organism to study symbiosis in the context of its association with its host, the Hawaiian bobtail squid Euprymna scolopes. The genetic tractability of this bacterium has facilitated the mapping of pathways that mediate interactions between these organisms. The protocols included here describe methods for genetic manipulation of V. fischeri. Following these protocols, the researcher will be able to introduce linear DNA via transformation to make chromosomal mutations, to introduce plasmid DNA via conjugation and subsequently eliminate unstable plasmids, to eliminate antibiotic resistance cassettes from the chromosome, and to randomly or specifically mutagenize V. fischeri with transposons. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Transformation of V. fischeri with linear DNA

Basic Protocol 2: Plasmid transfer into V. fischeri via conjugation

Support Protocol 1: Removing FRT-flanked antibiotic resistance cassettes from the V. fischeri genome

Support Protocol 2: Eliminating unstable plasmids from V. fischeri

Alternate Protocol 1: Introduction of exogenous DNA using a suicide plasmid

Alternate Protocol 2: Site-specific transposon insertion using a suicide plasmid

Alternate Protocol 3: Random transposon mutagenesis using a suicide plasmid

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费氏弧菌的基因操作

费氏弧菌是一种与致病性弧菌相关的非致病性生物。该细菌已被用作模式生物来研究其与宿主夏威夷短尾鱿鱼(Euprymna scolopes)之间的共生关系。这种细菌的遗传易感性促进了介导这些生物体之间相互作用的途径的绘制。这里包括的协议描述了费氏弧菌的遗传操作方法。根据这些方案，研究人员将能够通过转化引入线性DNA以产生染色体突变，通过偶联引入质粒DNA并随后消除不稳定的质粒，消除染色体上的抗生素抗性磁带，以及随机或特异性地用转座子诱变费氏弧菌。©2020 Wiley期刊有限责任公司基本方案1:用线性DNA转化V. fischeri基本方案2:通过偶联将质粒转移到V. fischeri支持方案1:从V. fischeri基因组中去除frt -侧翼抗生素耐药盒支持方案2:从V. fischeri中消除不稳定质粒替代方案1:使用自杀质粒引入外源DNA替代方案2:备选方案3:使用自杀质粒进行随机转座子突变

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 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.