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. Specifilcally, 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.
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.
- Streak out samples from glycerol stocks.
- Pick a single colony and grow up overnight 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
- 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
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