Feb 10, 2024

Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus (Sp) V.2

DOI
https://dx.doi.org/10.17504/protocols.io.x54v9dd1pg3e/v2
  • 1University of Massachusetts at Amherst
fritzlaylinlab.umass Fritz-Laylin
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DOI: https://dx.doi.org/10.17504/protocols.io.x54v9dd1pg3e/v2
External link: https://doi.org/10.1099/acmi.0.000566.v3
Collection Citation: Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin 2024. Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus (Sp). protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9dd1pg3e/v2Version created by fritzlaylinlab.umass Fritz-Laylin
Manuscript citation:
Edgar M Medina Kristyn A Robinson Kimberly Bellingham-Johnstun Giuseppe Ianiri Caroline Laplante Lillian K Fritz-Laylin Nicolas E Buchler (2020) Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology eLife 9:e52741. https://doi.org/10.7554/eLife.52741
License: This is an open access  collection  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: February 10, 2024
Last Modified: February 10, 2024
Collection  Integer ID: 94975
Keywords: Electroporation, Agrobacterium tumefaciens, Spizellomyces punctatus, chytrid fungi, chytrids, transformation of the chytrid fungus spizellomyces punctatus, chytrid fungus spizellomyces punctatus, transformation of the chytrid fungus, chytrid fungus, agrobacterium, mediated transformation, punctatus, sp
Funders Acknowledgements:
Gordon and Betty Moore Foundation
Grant ID: #9337
National Science Foundation
Grant ID: IOS-1827257
Pew Charitable Trusts
Grant ID: Pew Scholar award
Abstract
This is a collection of protocols for Agrobacterium-mediated transformation of the chytrid fungus
Spizellomyces punctatus.
Attachments
Icon for Spizellomyces_transformation_video.1.mp4
Spizellomyces_transf...
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Icon for Spizellomyces_transorfmation_video_transcript.pdf
Spizellomyces_transo...
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Icon for Transformation_protocol_outline_v2.pdf
Transformation_proto...
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Guidelines
Please thoroughly read through each protocol entry before starting, including the materials, guidelines, and warnings
Materials
Section 1: Dilute Salts Stock Solution I (10x)

  • 5 millimolar (mM) KH2PO4 (340.2 mg ) Potassium phosphate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0662-1KG
  • 5 millimolar (mM) KH2PO4 (435.5 mg ) Potassium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P3786-1KG
  • 5 millimolar (mM) (NH4)2HPO4 (330.15 mg ) Ammonium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #215996
  • 500 mL Water
  • Sterilize by filtration
  • Store at Room temperature for up to 12 months

Section 2: Dilute Salts Stock Solution II (10x)

  • 0.5 millimolar (mM) MgCl2 (11.9 mg ) 1 M Magnesium Chloride (MgCl2)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M8266
  • 0.5 millimolar (mM) CaCl2 (13.87 mg ) Calcium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C1016
  • 250 mL Water
  • Sterilize by filtration
  • Store at Room temperature for up to 12 months

Section 3: Dilute Salts Solution (1x) (Machlis, 1958)

  • 100 mL DS Stock Solution I
  • 100 mL DS Stock Solution II
  • 800 mL sterile water
  • Prepare solution in a sterile laminar flow hood with sterile supplies
  • Store at Room temperature for up to 12 months

Section 4: K1 Media (liquid and solid)

  • 0.06% Bacto Peptone (0.6 g ) (w/v; Bacto™ PeptoneThermo Fisher ScientificCatalog #211677 )
  • 0.04% Yeast Extracts (0.4 g ) (w/v; Fisher BioReagents™ Microbiology Media Additives: Yeast ExtractFisher ScientificCatalog #BP1422-2
  • 0.18% Glucose (1.8 g ) (w/v; D-( )-GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G5767-5KG
  • For solid media only: 1.5% (w/v) agar (15 g ) AgarFisher ScientificCatalog #BP1423-500
  • Water up to 1 L
  • Sterilize by autoclaving
  • Let cool to 60 °C before adding any selection antimicrobials
  • Store at 4 °C for up to 6 months

Section 5: LB media (liquid and solid)-- made from individual components

  • 1% Tryptone (10 g ) (w/v, TryptoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #T7293 )
  • 1% NaCl (10 g ) Sodium Chloride Fisher BioReagents™Fisher ScientificCatalog #BP358-1
  • 0.5% Yeast Extract (5 g ) (w/v; Fisher BioReagents™ Microbiology Media Additives: Yeast ExtractFisher ScientificCatalog #BP1422-2 )
  • For solid media only: 1.5% (w/v) agar (15 g ) AgarFisher ScientificCatalog #BP1423-500
  • Water up to 1 L
  • Sterilize by autoclaving
  • Let cool to 60 °C before adding any selection antimicrobials
  • Store at 4 °C for up to 6 months

Section 6: LB media (liquid and solid)-- commercially available

  • 25 g LB powder (LB Broth (Miller)Merck MilliporeSigma (Sigma-Aldrich)Catalog #L3522-1KG )
  • For solid media only: 1.5% agar (15 g ) (w/v; AgarFisher ScientificCatalog #BP1423-500 )
  • Water up to 1 L
  • Sterilize by autoclaving
  • Let cool to 60 °C before adding any selection antimicrobials
  • Store at 4 °C for up to 6 months

Section 7: Minimal Salts Solution (2.5x)

  • 26.6 millimolar (mM) KH2PO4 (3.625 g ) Potassium phosphate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0662-1KG
  • 29.4 millimolar (mM) KH2PO4 (5.125 g ) Potassium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P3786-1KG
  • 6.4 millimolar (mM) NaCl (0.375 g ) Sodium Chloride Fisher BioReagents™Fisher ScientificCatalog #BP358-1
  • 5 millimolar (mM) MgSO4·7H2O (1.250 g ) Magnesium sulfate heptahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #2303915
  • 1.1 millimolar (mM) CaCl2·2H2O (0.165 g ) Calcium Chloride DihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #C7902-500G
  • 22.3 micromolar (µM) FeSO4·7H2O (6.2 mg ) Iron(II) sulfate heptahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #F8263-1KG
  • 9.5 millimolar (mM) (NH4)2SO4 (1.250 g ) Ammonium sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A2939-500g
  • Water up to 1 L
  • No need to sterilize, precipitate is normal
  • Store at Room temperature for up to 1 year

Section 8: MES with acetosyringone

  • 40 Mass Percent MES 5.3 (7.7 g ) (2-(N-morpholino)ethanesulfonic acid)MES hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M2933-500G
  • 200 micromolar (µM) acetosyringone (0.0392 g ) 3′5′-Dimethoxy-4′-hydroxyacetophenone Merck MilliporeSigma (Sigma-Aldrich)Catalog #D134406-5G
  • pH with KOH
  • MES must be at 5.3 before adding acetosyringone
  • Water up to 50 mL after pHing
  • Filter sterilize, DO NOT autoclave
  • Add to IM recipe after other components are autoclaved and cooled

Section 9: Induction Media (liquid and solid)

  • 1x Minimal salts solution (400 mL of 2.5x stock solution, see recipe above)
  • 10 millimolar (mM) glucose (0.9 g ) (w/v; D-( )-GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G5767-5KG )
  • 0.5% glycerol (v/v; 5 mL ) Glycerol (Certified ACS) Fisher Chemical™Fisher ScientificCatalog #G33-1
  • For solid media only: 1.5% (w/v) agar (15 g ) AgarFisher ScientificCatalog #BP1423-500
  • Water up to 950 mL
  • Sterilize by autoclaving BEFORE adding MES with acetosyringone
  • 50 mL MES with acetosyringone (see recipe above; only add after autoclaving other components and cooling to 58 °C )
  • DO NOT autoclave acetosyringone, this will degrade the hormone
  • Store at 4 °C for up to 1 month
















































































Files
Protocol
Name
Protocol 1: Electroporation of Agrobacterium tumefaciens with a plasmid of interest
Version 1
Created by
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fritzlaylinlab.umass Fritz-LaylinUniversity of Massachusetts Amherst
Protocol
Name
Protocol 2: Culturing Spizellomyces punctatus (Sp) prior to transformation day
Version 1
Created by
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fritzlaylinlab.umass Fritz-LaylinUniversity of Massachusetts Amherst
Protocol
Name
Protocol 3: Growing liquid cultures of Agrobacterium prior to transformation day
Version 1
Created by
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fritzlaylinlab.umass Fritz-LaylinUniversity of Massachusetts Amherst
Protocol
Name
Protocol 4: Creating depressions in induction media plates
Version 1
Created by
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fritzlaylinlab.umass Fritz-LaylinUniversity of Massachusetts Amherst
Protocol
Name
Protocol 5: Agrobacterium-mediated transformation of Spizellomyces punctatus (Sp)
Version 1
Created by
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fritzlaylinlab.umass Fritz-LaylinUniversity of Massachusetts Amherst
Protocol
Name
Protocol 6: Selecting for Spizellomyces punctatus transformants
Version 1
Created by
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fritzlaylinlab.umass Fritz-LaylinUniversity of Massachusetts Amherst
Protocol
Name
Protocol 7: Picking colonies of transformed Spizellomyces punctatus (Sp)
Version 1
Created by
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fritzlaylinlab.umass Fritz-LaylinUniversity of Massachusetts Amherst