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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

MF Michael B. Francis
JS Joseph A. Sorg

发布: 2013年12月20日第3卷第24期 DOI: 10.21769/BioProtoc.1001 浏览次数: 11642

评审: Anonymous reviewer(s)

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The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

May 2013

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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) search Germination (发芽) search Spore (孢子) search Mutagenesis (诱变) search

Materials and Reagents

  1. A petri plate of viable C. difficile cells
  2. Ethyl methanesulfonate (EMS)
  3. Rifampin
  4. Sterile water
  5. Sucrose
  6. 10% (w/v) taurocholic acid
  7. Thioglycollate
  8. Lysozyme
  9. Brain Heart Infusion (BD BactoTM, catalog number: 237500 )
  10. Yeast Extract (BD DifcoTM, catalog number: 212750 )
  11. 0.1% (w/v) L-cysteine (Sigma-Aldrich, catalog number: C7352 )
  12. BHIS medium (see Recipes)
  13. BHIS agar medium (see Recipes)

Equipment

  1. 15 ml conical screw cap tubes
  2. 50 ml conical screw cap tubes
  3. Anaerobic chamber
  4. Spectrophotometer or microplate reader
  5. Biosafety cabinet
  6. Bench-top microcentrifuge
  7. Swinging-bucket centrifuge
  8. 65 °C heat block
  9. 50 °C water bath
  10. 37 °C water bath

Procedure

English
中文翻译

文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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

  1. 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.
  2. 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.

    3. ris ris Download Citation in RIS Format

分类

微生物学 > 微生物遗传学 > 诱/突变

微生物学 > 微生物细胞生物学 > 细胞分离和培养

分子生物学 > DNA > 诱/突变

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