EMS诱变鉴定艰难梭菌萌发无效显型菌株

Michael  B.  Francis; Joseph  A.  Sorg

doi:10.21769/BioProtoc.1001

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Peer-reviewed

EMS Mutagenesis of Clostridium difficile to Identify Strains with Germination-null Phenotypes

EMS诱变鉴定艰难梭菌萌发无效显型菌株

M Michael B. Francis

JS Joseph A. Sorg email

发布: 2013年12月20日第3卷第24期 DOI: 10.21769/BioProtoc.1001 浏览次数: 11219

评审: Anonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

May 2013

实验方案合集

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Abstract

Clostridium difficile is a Gram-positive, spore-forming, strict anaerobe and the leading cause of antibiotic-associated diarrhea (McFarland, 2008). Germination by C. difficile spores is the first step in pathogenesis. Thus, identifying mechanisms of C. difficile spore germination may lead to novel anti-germination therapies. This protocol describes a method for identifying germination-null phenotypes for C. difficile spores by introducing random mutations into actively growing C. difficile using ethyl methanesulfonate (EMS) as a mutagen (Francis et al., 2013).

Keywords: Clostridium difficile (难辨梭状芽孢杆菌)

Germination (发芽)

Spore (孢子)

Mutagenesis (诱变)

Materials and Reagents

A petri plate of viable C. difficile cells
Ethyl methanesulfonate (EMS)
Rifampin
Sterile water
Sucrose
10% (w/v) taurocholic acid
Thioglycollate
Lysozyme
Brain Heart Infusion (BD Bacto^TM, catalog number: 237500 )
Yeast Extract (BD Difco^TM, catalog number: 212750 )
0.1% (w/v) L-cysteine (Sigma-Aldrich, catalog number: C7352 )
BHIS medium (see Recipes)
BHIS agar medium (see Recipes)

Equipment

15 ml conical screw cap tubes
50 ml conical screw cap tubes
Anaerobic chamber
Spectrophotometer or microplate reader
Biosafety cabinet
Bench-top microcentrifuge
Swinging-bucket centrifuge
65 °C heat block
50 °C water bath
37 °C water bath

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Francis, M. B. and Sorg, J. A. (2013). EMS Mutagenesis of Clostridium difficile to Identify Strains with Germination-null Phenotypes. Bio-protocol 3(24): e1001. DOI: 10.21769/BioProtoc.1001.
Francis, M. B., Allen, C. A., Shrestha, R. and Sorg, J. A. (2013). Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection. PLoS Pathog 9(5): e1003356.