Mushroom Genetic Engineering Toolkit v0.01

Mushroom Genetic Engineering Toolkit v0.01

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What is the toolkit

Mushroom-forming fungi are important ecological and commercial organisms. In nature, mushroom-forming fungi are critical to Earth’s carbon cycle. Humans revere mushrooms for food, medicinal and spiritual practices. Mushrooms emerge from a fungal organism’s vegetative root-like structure, mycelium, which are increasingly being investigated for their material properties. The food, medicine and material properties of mushrooms are directly affected by their genetic makeup. Therefore, tools for exploring their genetic potential would benefit ecological and commercial mushroom research.

This experimental toolkit for genetic engineering mushrooms contains three plasmids for polyethylene glycol-mediated transformation (PMT) and one plasmid for Agrobacterium tumefaciens-mediated transformation (ATMT).

By "experimental" we mean that the plasmids may or may not work. Specifically, all plasmids in the set have had all BsaI restriction sites removed. All plasmids have been sequence-confirmed and have been confirmed to replicate in E. coli. One of the plasmids, pOMRP4β, has been confirmed to replicate in A. tumefaciens but ATMT of G. lucidum with pOMRP4β has not been confirmed. Proper function of the G. lucidum-specific expression cassettes in all plasmids has also not been confirmed.

 

Technical details

The set contains four genes of interest: three that have been codon-optimized for expression in G. lucidum and one mutated native gene, as follows: pOMRP1 contains hygromycin B phosphotransferase (hph), pOMRP2 contains free-use Green Fluorescent Protein (fuGFP), pOMRP3 contains nanoLuciferase (nanoLuc), and pOMRP4β, a derivative of pCAMBIA1300 that has been modified to contain two genes of interest, a mutated iron-sulfur subunit of the succinate dehydrogenase (SdhB) gene from G. lucidum containing a point mutation—referred to as (CbxR)—that has been reported to confer resistance to carboxin, and hph. fuGFP, hph, and nanoLuc are regulated by the putative promoter and terminator of the G. lucidum 10597 SS1 glyceraldehyde-3-phosphate dehydrogenase (GPD) gene. CbxR is regulated by the native promoter and terminator. See the plasmid maps for further information.

Future iterations will make all components in all of the plasmids in the collection MoClo compatible.

 

What can these plasmids be used for

The experimental toolkit is designed to be used to transform the mushroom G. lucidum 10597 SS1 but it may be effective for transforming other fungi. Users should be aware that the proper functioning of the associated plasmids has not been confirmed. pOMRP1, pOMRP2 and pOMRP3 are designed to be used in conjunction with PMT, electroporation, biolistic transformation, shock-wave-mediated transformation or other non-ATMT methods. pOMRP4β is designed to be used for ATMT of G. lucidum 10597 SS1 by way of A. tumefaciens strain AGL-1.

 

Please Note: The Mushroom Genetic Engineering Toolkit is available in two formats: E. coli glycerol stocks and purified nucleic acids. Please specify in the order notes if you need a particular format.


Sample Workflow

  1. Streak out samples from glycerol stocks.
  2. Pick a single colony and grow up overnight for maxi- or mini-prep.
  3. Harvest the overnight culture and prepare the plasmid.
  4. Choose a transformation method corresponding to the plasmid-type (PMT or ATMT).
    1. For PMT:
      1. Grow up G. lucidum and produce protoplasts. Experimental protocol: https://openwetware.org/wiki/Protoplast_Production
      2. Transform G. lucidum. Experimental protocol: https://openwetware.org/wiki/Plasmid_delivery_using_PEG-mediated_transformation
    2. For ATMT:
      1. Transform the plasmid into A. tumefaciens. Experimental protocol: https://openwetware.org/wiki/Biomaterials:_Culture_Samples,_Plasmids,_Agrobacterium_strains
      2. Follow the protocol according to Transformation of the mycorrhizal fungus Laccaria bicolor using Agrobacterium tumefaciens. Kemppainen, M., Pardo, A. Bioengineered Bugs. 2011. https://doi.org/10.4161/bbug.2.1.14394
      3. Or, the experimental protocol: https://openwetware.org/wiki/Plate-based_Agrobacterium-mediated_transformation


Where can I find more information

 

Genes

Gene Name Freegenes ID
pOMRP4β sdhB_hph_Gluc BBF10K_000571
pOMRP1 hph_Gluc BBF10K_000572
pOMRP2 fuGFP_Gluc BBF10K_000573
pOMRP3 NanoLuc_Gluc BBF10K_000574

Download all of this information as a CSV from our GitHub.

 

Bionet

The bionet enables open peer-peer exchange of functional biomaterials and associated data. This product may also be available from bionet nodes that are more convenient to you. Here are other bionet nodes who may be willing to provide you this specific product.

Name Contact Country
Britt Holick Katiehoffmanneuro {at} yahoo {dot} com United States
Jordan Gonzalez jgonzalez {at} thecitizensciencelab {dot} org United States
Brian Eberhardt thesporehouse {at} gmail {dot} com United States
Majed Alghamdi mafa2 {at} le {dot} ac {dot} uk United Kingdom
Ahmad Suparmin ahmad.suparmin {at} ugm {dot} ac {dot} id Indonesia
Ery Fukushima ery.fukushima {at} ikiam {dot} edu {dot} ec Ecuador
Radames Cordero rcorder4 {at} jhu {dot} edu United States
Swaraj Kunal swaraj.avisa {at} gmail {dot} com India
Vasilis Joseph vassilisjoseph {at} gmail {dot} com Cyprus
Javier Villa Javier {at} verdosome {dot} com United States
Peter Voyvodic cbs.cnrs.fr France
Lucas Morelli Lmorellip {at} gmail {dot} com Brazil
Jose David Rosales jdrr55 {at} yahoo {dot} com https://www {dot} linkedin {dot} com/in/david-rosales-574a7151/ Venezuela
robson tramontina robson.tramontina {at} gmail {dot} com Brazil
Rachel Roberts NA United States
Xavier Coadic xcoadic {at} protonmail {dot} com France
WILLIAN COSTA willianbarelac {at} gmail {dot} com Brazil
John Grossman Mushmitch {at} yahoo {dot} com United States

Download all of this information as a CSV from our GitHub.

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