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 shipped as purified and dried down plasmid DNA stained with Cresol Red in one 96-well plate. Each well contains approximately 50 ng of DNA. Cresol Red will not impact plasmid transformation.

pOMRP4β (BBF10K_000571) has been found to contain an insert of 1338 bp. This insert is not in the middle of any genes.

Sample Workflow

Transform into E. coli Top10 cells or equivalent.
Pick a single colony and culture overnight at 37C for maxi- or mini-prep.
Harvest the overnight culture and prepare the plasmid.
Choose a transformation method corresponding to the plasmid-type (PMT or ATMT).

For PMT:

Grow up G. lucidum and produce protoplasts. Experimental protocol: https://openwetware.org/wiki/Protoplast_Production
Transform G. lucidum. Experimental protocol: https://openwetware.org/wiki/Plasmid_delivery_using_PEG-mediated_transformation

For ATMT:

Transform the plasmid into A. tumefaciens. Experimental protocol: https://openwetware.org/wiki/Biomaterials:_Culture_Samples,_Plasmids,_Agrobacterium_strains
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
Or, the experimental protocol: https://openwetware.org/wiki/Plate-based_Agrobacterium-mediated_transformation

Where can I find more information

Platemap
Original research article describing the use of PMT, ATMT, hph, CbxR, and eGFP with G. lucidum.

Genetic engineering of Ganoderma lucidum for the efficient production of ganoderic acids. Jun-Wei Xu and Jian-Jiang Zhong. Bioengineered. 2015. https://dx.doi.org/10.1080%2F21655979.2015.1119341

Original research article describing the pCAMBIA1300 backbone of pOMRP4β.

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

News article and Addgene website link describing Free Use GFP.

Small Things Considered: The Story of Free Use GFP. https://schaechter.asmblog.org/schaechter/2019/05/the-story-of-free-use-gfp-fugfp.html
Addgene website link for fuGFP: https://www.addgene.org/127674/

Endy Lab Benchling links to plasmid maps:

pOMRP1 - https://benchling.com/s/seq-XmkA8WbTkcqSYCmcOqRX
pOMRP2 - https://benchling.com/s/seq-DYH7s0kKrSWI9YXQz0GA
pOMRP3 - https://benchling.com/s/seq-mPB6jIcWcfkBhPA9F0Dv
pOMRP4β - https://benchling.com/s/seq-DL7qaMldpB5CvriNCxs6

Teaser image source

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
Kyle Erlenbeck	krerlenbeck {at} dcsdk12 {dot} org	United States
Piero Beraun	piero.beraun {at} utec {dot} edu {dot} pe	Italy
Ery Fukushima	ery.fukushima {at} ikiam {dot} edu {dot} ec	Ecuador
Mikhail Khvochtchev	mikhail.khv {at} mahidol {dot} edu	Thailand
Xavier Coadic	xcoadic {at} protonmail {dot} com	France
Nadia Odaliz Chamana Chura	nadia.chamana {at} utec {dot} edu {dot} pe	France
Ahmad Suparmin	ahmad.suparmin {at} ugm {dot} ac {dot} id	Indonesia
Emiliano Emiliano	emilianovilardo {at} gmail {dot} com	Argentina
Haoyong WANG	research	China
Jimmy Gledson Hayden Linhares	haydenlinhars {at} gmail {dot} com	Brazil
Jordan Gonzalez	jgonzalez {at} thecitizensciencelab {dot} org	United States
Benjamin Arias	barias {at} alumni {dot} usfq {dot} edu {dot} ec	Ecuador
Vasilis Joseph	vassilisjoseph {at} gmail {dot} com	Cyprus
Emilia Mazza	emilia {at} michroma {dot} co	Argentina
Rachel Roberts	NA	United States
John Grossman	NA	United States
Simon Vandelook	Simon.vandelook {at} vub {dot} be	Belgium
Majed Alghamdi	mafa2 {at} le {dot} ac {dot} uk	United Kingdom
WILLIAN COSTA	willianbarelac {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
Moritz Venne	moritz.venne {at} web {dot} de	Germany
Lucas Morelli	Lmorellip {at} gmail {dot} com	Brazil
Swaraj Kunal	swaraj.avisa {at} gmail {dot} com	India
Hernán Rebolledo	lenaranjo {at} gmail {dot} com	United States
Britt Holick	Katiehoffmanneuro {at} yahoo {dot} com	United States
mehmet tardu	mtardu {at} gmail {dot} com	Turkey
John Grossman	Mushmitch {at} yahoo {dot} com	United States
Javier Villa	Javier {at} verdosome {dot} com	United States
Brian Eberhardt	thesporehouse {at} gmail {dot} com	United States
Peter Voyvodic	cbs.cnrs.fr	France
Radames Cordero	rcorder4 {at} jhu {dot} edu	United States

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

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