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Team:Cornell/project/background/fungal genetic engineering
From 2013.igem.org
Fungal Genetic Engineering
Recent years have shown increasing interest in the genetic engineering of filamentous fungi. With this interest has come many developments in transformation methods. Currently, there are two main methods commonly used to transform fungal species with recombinant DNA: Agrobacterium mediated transformation and protoplasting [1].
Agrobacterium tumefaciens mediated transformation (AMT) is a method by which Agrobacterium are utilized to transfect fungal cells [3]. Genetic constructs are first transformed into the T-DNA of A. tumefaciens. A. tumefaciens, when co-cultured with fungi, will then transfect the fungal cells, inserting the gene of interest into the fungal genetic material. This DNA can then be incorporated into the genome through random insertions or homologous recombination.
Cochliobolus heterostrophus
The more common method, however, involves protoplasting. Protoplasts are formed by using commercially available enzymes to remove the wall from fungal cells. Once these fragile cells are formed, they can be transformed with polyethylene glycol (PEG). By exposing the fragile protoplasts to PEG and recombinant DNA the cells uptake the DNA. Integration of the DNA into the fungal genome can then occur via homologous recombination, random insertion, or restriction enzyme mediated integration (REMI) [2]. Typically, homologous recombination is the most efficient, though this varies by species. Transformation markers are typically used in order to select for transformants. In fungal strains, these typically include resistances for hygromycin and geneticin.
With both Agrobacterium mediated transformation and protoplasting, selection markers are used to select successful transformants. Two commonly used selection markers are the antibiotics hygromycin and geneticin, which are both effective against the strains used in our project: Ganoderma lucidum and Cochliobolus heterostrophus.
Protoplasting of both Ganoderma lucidum and Cochliobolus heterostrophus was attempted but has thus far only been successful with Cochliobolus heterostrophus. The enzymes typically used for protoplasting Ganoderma lucidum are exclusively available in China, so we attempted to use alternate enzymes unsuccessfully [4]. The Cochliobolus protoplasts were then transformed with various constructs. Because protoplasting of Ganoderma lucidum was unsuccessful, we also began preliminary work to transform Ganoderma lucidum with Agrobacterium mediated integration.
With both Agrobacterium mediated transformation and protoplasting, selection markers are used to select successful transformants. Two commonly used selection markers are the antibiotics hygromycin and geneticin, which are both effective against the strains used in our project: Ganoderma lucidum and Cochliobolus heterostrophus.
Protoplasting of both Ganoderma lucidum and Cochliobolus heterostrophus was attempted but has thus far only been successful with Cochliobolus heterostrophus. The enzymes typically used for protoplasting Ganoderma lucidum are exclusively available in China, so we attempted to use alternate enzymes unsuccessfully [4]. The Cochliobolus protoplasts were then transformed with various constructs. Because protoplasting of Ganoderma lucidum was unsuccessful, we also began preliminary work to transform Ganoderma lucidum with Agrobacterium mediated integration.
References
1. Weld, R. J., Plummer, K. M., Carpenter, M. A., & Ridgeway, H. J. (n.d.). Appraoches to functional genomics in filamentous fungi. N.p.: Nature.2. Turgeon, G. B., Condon, B., Liu, J., & Zhang, N. (2010). Protoplast Transformation of Filamentous Fungi (Vol. 638). N.p.: Molecular and Cell Biology for Fungi.
3. Feldmann, K. A., & Marks, D. M. (1986). Agrobacterium-mediated transformation of germinating seeds of Arabidopsi thaliana: A non-tissue culture approach. N.p.: Zoecon Research Institute.
4. Sun, L., Cai, H., Xu, W., Hu, Y., Gao, Y., & Lin, Z. (2001). Efficient Transformation of The Medicinal Mushroom Ganoderma Lucidum (19th ed.). N.p.: Plant Molecular Biology Reporter.
