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Peer-reviewed

Polyethylene Glycol-mediated Transformation of Drechmeria coniospora

聚乙二醇介导的圆锥掘氏梅里霉的转化

LH Le D. He
JE Jonathan J. Ewbank

发布: 2017年03月05日第7卷第5期 DOI: 10.21769/BioProtoc.2157 浏览次数: 8914

评审: Arsalan DaudiMichael TschernerAksiniya Asenova

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Abstract

Drechmeria coniospora is a nematophagous fungus and potential biocontrol agent. It belongs to the Ascomycota. It is related to Hirsutella minnesotensis, another nematophagous fungus but, phylogenetically, it is currently closest to the truffle parasite Tolypocladium ophioglossoides. Together with its natural host, Caenorhabditis elegans, it is used to study host-pathogen interactions. Here, we report a polyethylene glycol-mediated transformation method (Turgeon et al., 2010; Ochman et al., 1988) for this fungus. The protocol can be used to generate both knock-in or knock-out strains (Lebrigand et al., 2016).

Keywords: Fungus (真菌) search Genetic engineering (基因工程) search Nematophagous (食线虫) search Pathogen (病原体) search Caenorhabditis elegans (秀丽隐杆线虫) search

Background

D. coniospora has been developed as a model pathogen for the study of innate immunity in C. elegans (Lebrigand et al., 2016 and references therein). D. coniospora grows slowly on standard growth media, rendering in vitro study difficult and making it hard to develop transformation methods. We report here a culture method that allows the rapid production of large quantities of D. coniospora, opening the way to its genetic modification. Polyethylene glycol-mediated transformation is probably the simplest method that has been broadly applied to modify fungi. We found that it can be used with D. coniospora, thus providing the first approach to modify its genome deliberately.

Materials and Reagents

  1. Pasteur pipettes (VWR, catalog number: 6121701 )
  2. Eppendorf tube 1.5 ml (Sigma-Aldrich, catalog: T9661 )
  3. Petri dish (Greiner Bio one, catalog number: 633185 )
  4. 50 ml conical tube (Corning, Falcon®, catalog number: 352070 )
  5. 15 ml conical tube (Corning, Falcon®, catalog number: 352196 )
  6. 14 ml polystyrene round bottom tube (Corning, Falcon®, catalog number: 352051 )
  7. Miracloth (EMD Millipore, catalog number: 475855 )
  8. Syringe 25 ml (Terumo, catalog number: SS-20ES )
  9. Acrodisc® syringe filters (Pall, catalog number: 4187 )
  10. Aluminum foil (Fisher Scientific, catalog number: 01213102 )
  11. Escherichia coli strain OP50
  12. Caenorhabditis elegans strain N2
  13. Drechmeria coniospora recipient strain ATCC 96282
  14. pLH4237 plasmid DNA, contains a hygromycin B phosphotransferase::GFP chimeric gene driven by D. coniospora β-tubulin promoter (β-tubp::HPH::GFP, Lebrigand et al., 2016)
  15. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518 )
  16. Gentamicin solution (Sigma-Aldrich, catalog number: G1272 )
  17. Hygromycin B (Thermo Fisher scientific, GibcoTM, catalog number: 10687010 )
  18. di-Potassium hydrogen orthophosphate, K2HPO4 (VWR, catalog number: 26931.263 )
  19. Potassium dihydrogen phosphate, KH2PO4 (VWR, catalog number: 0781-1KG )
  20. Bacto agar (BD, Bacto, catalog number: 214010 )
  21. Bacto peptone (BD, Bacto, catalog number: 211677 )
  22. Sodium chloride, NaCl (VWR, catalog number: 27810.295 )
  23. Cholesterol (Sigma-Aldrich, catalog number: C3045 )
  24. Ethanol (VWR, catalog number: 20821.321 )
  25. Magnesium sulfate, MgSO4 (Sigma-Aldrich, catalog number: M7506 )
  26. Calcium chloride, CaCl2 (Sigma-Aldrich, catalog number: C1016 )
  27. Yeast extract (Douchefa Biochemie, catalog number: Y1333 )
  28. D-sorbitol (Sigma-Aldrich, catalog number: S3889 )
  29. Aurintricarboxylic acid, ATA (EMD Millipore, catalog number: 189400 )
  30. Tris/HCl (Sigma-Aldrich, catalog number: T6666 )
  31. Polyethylene glycol (PEG 3350) (Sigma-Aldrich, catalog number: P4338 )
  32. Caylase C4 (CAYLA, catalog number: caseC4-5 ) Alternative can be lysing enzymes (Sigma-Aldrich, catalog number: L1412)
    Note: The hyperlink for caseC4-5 is not available anymore. Alternative could be lysing enzyme, and we are still testing its efficiency in our lab.
  33. Phosphate buffer (see Recipes)
  34. Medium (see Recipes)
    1. NGM-AG
    2. NGMY
    3. NGMY liquid
    4. NGMS
  35. 15 mM ATA solution (see Recipes)
  36. 50 mM NaCl solution (see Recipes)
  37. CaCl2 1700 (see Recipes)
  38. Lysis buffer (see Recipes)
  39. STC 1700 (see Recipes)
  40. PEG 1700 (see Recipes)

Equipment

  1. Stereomicroscope (Leica Microsystems, model: MZ16 F )
  2. HERAsafe KS safety cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: HerasafeTM KS Class II , catalog number: 51022751)
  3. Centrifuge (Eppendorf, catalog number: 5810 R )
  4. Funnel
  5. Spatula
  6. Cloth-plugged 250 ml glass Erlenmeyer flask
  7. Rotary shaker for culture flasks (Eppendorf, model: New Brunswick Scientific Innova 4080 )
  8. Counting chambers (Burker, Tiefe 0.1000 m, 0.0025 mm2)
  9. Balancer
  10. Pipetman P1000
  11. autoclave

Procedure

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版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

He, L. D. and Ewbank, J. J. (2017). Polyethylene Glycol-mediated Transformation of Drechmeria coniospora. Bio-protocol 7(5): e2157. DOI: 10.21769/BioProtoc.2157.

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分类

微生物学 > 微生物遗传学 > 转化

分子生物学 > DNA > 转化

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