Feb 09, 2024

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

DOI
dx.doi.org/10.17504/protocols.io.x54v9dd1pg3e/v2
  • 1University of Massachusetts at Amherst
fritzlaylinlab.umass Fritz-Laylin
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DOI: 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 09, 2024
Last Modified: February 09, 2024
Collection Integer ID: 94975
Keywords: Electroporation, Agrobacterium tumefaciens, Spizellomyces punctatus, chytrid fungi, chytrids
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...
912.1MB
Icon for Spizellomyces_transorfmation_video_transcript.pdf
Spizellomyces_transo...
79KB
Icon for Transformation_protocol_outline_v2.pdf
Transformation_proto...
189KB
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)

  • Concentration5 millimolar (mM) KH2PO4 (Amount340.2 mg ) ReagentPotassium phosphate monobasicSigma AldrichCatalog #P0662-1KG
  • Concentration5 millimolar (mM) KH2PO4 (Amount435.5 mg ) ReagentPotassium phosphate dibasicSigma AldrichCatalog #P3786-1KG
  • Concentration5 millimolar (mM) (NH4)2HPO4 (Amount330.15 mg ) ReagentAmmonium phosphate dibasicSigma AldrichCatalog #215996
  • 500 mL Water
  • Sterilize by filtration
  • Store at TemperatureRoom temperature for up to 12 months

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

  • Concentration0.5 millimolar (mM) MgCl2 (Amount11.9 mg ) Reagent 1 M Magnesium Chloride (MgCl2)Sigma AldrichCatalog #M8266
  • Concentration0.5 millimolar (mM) CaCl2 (Amount13.87 mg ) ReagentCalcium chlorideSigma – AldrichCatalog #C1016
  • Amount250 mL Water
  • Sterilize by filtration
  • Store at TemperatureRoom temperature for up to 12 months

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

  • Amount100 mL DS Stock Solution I
  • Amount100 mL DS Stock Solution II
  • Amount800 mL sterile water
  • Prepare solution in a sterile laminar flow hood with sterile supplies
  • Store at TemperatureRoom temperature for up to 12 months

Section 4: K1 Media (liquid and solid)

  • 0.06% Bacto Peptone (Amount0.6 g ) (w/v; ReagentBacto™ PeptoneThermo Fisher ScientificCatalog #211677 )
  • 0.04% Yeast Extracts (Amount0.4 g ) (w/v; ReagentFisher BioReagents™ Microbiology Media Additives: Yeast ExtractFisher ScientificCatalog #BP1422-2
  • 0.18% Glucose (Amount1.8 g ) (w/v; ReagentD-( )-GlucoseMillipore SigmaCatalog #G5767-5KG
  • For solid media only: 1.5% (w/v) agar (Amount15 g ) ReagentAgarFisher ScientificCatalog #BP1423-500
  • Water up to Amount1 L
  • Sterilize by autoclaving
  • Let cool to Temperature60 °C before adding any selection antimicrobials
  • Store at Temperature4 °C for up to 6 months

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

  • 1% Tryptone (Amount10 g ) (w/v, ReagentTryptoneMillipore SigmaCatalog #T7293 )
  • 1% NaCl (Amount10 g ) ReagentSodium Chloride Fisher BioReagents™Fisher ScientificCatalog #BP358-1
  • 0.5% Yeast Extract (Amount5 g ) (w/v; ReagentFisher BioReagents™ Microbiology Media Additives: Yeast ExtractFisher ScientificCatalog #BP1422-2 )
  • For solid media only: 1.5% (w/v) agar (Amount15 g ) ReagentAgarFisher ScientificCatalog #BP1423-500
  • Water up to Amount1 L
  • Sterilize by autoclaving
  • Let cool to Temperature60 °C before adding any selection antimicrobials
  • Store at Temperature4 °C for up to 6 months

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

  • Amount25 g LB powder (ReagentLB Broth (Miller)Millipore SigmaCatalog #L3522-1KG )
  • For solid media only: 1.5% agar (Amount15 g ) (w/v; ReagentAgarFisher ScientificCatalog #BP1423-500 )
  • Water up to Amount1 L
  • Sterilize by autoclaving
  • Let cool to Temperature60 °C before adding any selection antimicrobials
  • Store at Temperature4 °C for up to 6 months

Section 7: Minimal Salts Solution (2.5x)

  • Concentration26.6 millimolar (mM) KH2PO4 (Amount3.625 g ) ReagentPotassium phosphate monobasicSigma AldrichCatalog #P0662-1KG
  • Concentration29.4 millimolar (mM) KH2PO4 (Amount5.125 g ) ReagentPotassium phosphate dibasicSigma AldrichCatalog #P3786-1KG
  • Concentration6.4 millimolar (mM) NaCl (Amount0.375 g ) ReagentSodium Chloride Fisher BioReagents™Fisher ScientificCatalog #BP358-1
  • Concentration5 millimolar (mM) MgSO4·7H2O (Amount1.250 g ) ReagentMagnesium sulfate heptahydrateMillipore SigmaCatalog #2303915
  • Concentration1.1 millimolar (mM) CaCl2·2H2O (Amount0.165 g ) ReagentCalcium Chloride DihydrateSigmaCatalog #C7902-500G
  • Concentration22.3 micromolar (µM) FeSO4·7H2O (Amount6.2 mg ) ReagentIron(II) sulfate heptahydrateMillipore SigmaCatalog #F8263-1KG
  • Concentration9.5 millimolar (mM) (NH4)2SO4 (Amount1.250 g ) ReagentAmmonium sulfateMillipore SigmaCatalog #A2939-500g
  • Water up to Amount1 L
  • No need to sterilize, precipitate is normal
  • Store at TemperatureRoom temperature for up to 1 year

Section 8: MES with acetosyringone

  • Concentration40 Molarity (M) MES Ph5.3 (Amount7.7 g ) (2-(N-morpholino)ethanesulfonic acid)ReagentMES hydrateMillipore SigmaCatalog #M2933-500G
  • Concentration200 micromolar (µM) acetosyringone (Amount0.0392 g ) Reagent3′5′-Dimethoxy-4′-hydroxyacetophenone Sigma AldrichCatalog #D134406-5G
  • pH with KOH
  • MES must be at Ph5.3 before adding acetosyringone
  • Water up to Amount50 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 (Amount400 mL of 2.5x stock solution, see recipe above)
  • Concentration10 millimolar (mM) glucose (Amount0.9 g ) (w/v; ReagentD-( )-GlucoseMillipore SigmaCatalog #G5767-5KG )
  • 0.5% glycerol (v/v; Amount5 mL ) ReagentGlycerol (Certified ACS) Fisher Chemical™Fisher ScientificCatalog #G33-1
  • For solid media only: 1.5% (w/v) agar (Amount15 g ) ReagentAgarFisher ScientificCatalog #BP1423-500
  • Water up to Amount950 mL
  • Sterilize by autoclaving BEFORE adding MES with acetosyringone
  • Amount50 mL MES with acetosyringone (see recipe above; only add after autoclaving other components and cooling to Temperature58 °C )
  • DO NOT autoclave acetosyringone, this will degrade the hormone
  • Store at Temperature4 °C for up to 1 month
















































































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