Yersinia pseudotuberculosis: Cultivation, Storage, and Methods for Introducing DNA

Current Protocols in Microbiology Pub Date : 2020-10-20 DOI:10.1002/cpmc.122

Robert K. Davidson, Kimberly M. Davis

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引用次数: 7

Abstract

Yersinia pseudotuberculosis has been studied for many decades, and research on this microbe has taught us a great deal about host-pathogen interactions, bacterial manipulation of host cells, virulence factors, and the evolution of pathogens. This microbe should not be cultivated at 37°C because this is a trigger that the bacterium uses to sense its presence within a mammalian host and results in expression of genes necessary to colonize a mammalian host. Prolonged growth at this temperature can result in accumulation of mutations that reduce the virulence of the strain, so all protocols need to be modified for growth at room temperature, or 26°C. This article describes protocols for cultivating this microbe and for its long-term storage and its genetic manipulation by transformation and conjugation. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Growth of Y. pseudotuberculosis from a stock

Basic Protocol 2: Growth of Y. pseudotuberculosis in liquid medium from a single colony

Basic Protocol 3: Freezing Y. pseudotuberculosis in glycerol for long-term storage

Basic Protocol 4: Transformation of Y. pseudotuberculosis by electroporation

Basic Protocol 5: Tri-parental mating/conjugation

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假结核耶尔森菌:培养、储存和DNA导入方法

假结核耶尔森菌已经被研究了几十年，对这种微生物的研究教会了我们很多关于宿主-病原体相互作用、细菌对宿主细胞的操纵、毒力因子和病原体进化的知识。这种微生物不应该在37°C下培养，因为这是细菌用来感知其在哺乳动物宿主内存在的触发因素，并导致在哺乳动物宿主中定植所需的基因表达。在此温度下的长时间生长可能导致突变的积累，从而降低菌株的毒力，因此所有方案都需要修改，以便在室温或26°C下生长。本文介绍了这种微生物的培养、长期储存以及通过转化和偶联进行遗传操作的方案。©2020 Wiley期刊有限公司。基本方案1:从储存中培养假结核耶氏菌。基本方案2:在单个菌落的液体培养基中培养假结核耶氏菌。基本方案3:在甘油中冷冻假结核耶氏菌以长期储存。基本方案4:电孔法转化假结核耶氏菌。基本方案5:三亲本交配/偶联

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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.