Team:Cornell/project/background/fungal genetic engineering

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<h2 class="centered">Fungal Genetic Engineering</h2>
<h2 class="centered">Fungal Genetic Engineering</h2>
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Recent years have shown increasing interest in the genetic engineering of filamentous fungus. This interest has been supplemented by many developments of transformation systems. Currently, there are 3 main methods commonly used to transform fungal species with recombinant DNA[4]. The primary method is PEG transformation, whereby the fungal species is protoplasted to rid the cells of their cell wall. By exposing the fragile protoplasts to polyethylene glycol 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)[3]. Typically homologous recombination is the most efficient. This does vary by species however. Transformation markers are typically used in order to select for transformants. In fungal strains these typically include resistances for hygromycin and geniticin. <br>
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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: <i>Agrobacterium</i> mediated transformation and protoplasting [1].
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Agrobacterium mediated transformation(AMT) is a method by which the fungus is co-cultivated with agrobacterium[1]. The agrobacterium is cloned to carry specific plasmids and uptakes through horizontal gene transfer, the fungus integrates the plasmids of the agrobacterium. <br>
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Ganoderma lucidum and Cochliobolus heterostrophus were used as the fungal strains used for genetic engineering. Protoplasting on both organisms was attempted but was only successful with Cochliobolus heterostrophus. The protoplasts were then transformed with various constructs. The enzymes typically used for protoplasting Ganoderma lucidum are exclusively available in china and thus alternate enzymes were attempted[2]. Future attempts at transforming Ganoderma lucidum will be done with agrobacterium mediated integration.
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<h3>Reference</h3>
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1. Feldmann, K. A., & Marks, D. M. (1986). <i> Agrobacterium-mediated transformation of germinating seeds of Arabidopsi thaliana: A non-tissue culture approach.</i> N.p.: Zoecon Research Institute. <br>
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<i>Agrobacterium tumefaciens</i> mediated transformation (AMT) is a method by which <i>Agrobacterium</i> are utilized to transfect fungal cells [3]. Genetic constructs are first transformed into the T-DNA of <i>A. tumefaciens</i>. <i>A. tumefaciens</i>, 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.
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2. Sun, L., Cai, H., Xu, W., Hu, Y., Gao, Y., & Lin, Z. (2001). <i> Efficient Transformation of The Medicinal Mushroom Ganoderma Lucidum (19th ed.). N.p.: Plant Molecular Biology Reporter.</i> <br>
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                              <i>Cochliobolus heterostrophus</i>
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3. Turgeon, G. B., Condon, B., Liu, J., & Zhang`, N. (2010). <i>Protoplast Transformation of Filamentous Fungi (Vol. 638). N.p.: Molecular and Cell Biology for Fungi.</i> <br>
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The more common method, however, involves protoplasting. Protoplasts are formed 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.  
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4. Weld, R. J., Plummer, K. M., Carpenter, M. A., & Ridgeway, H. J. (n.d.). <i>Appraoches to functional genomics in filamentous fungi. N.p.: Nature.</i> <br>
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With both <i>Agrobacterium</i> 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: <i>Ganoderma lucidum</i> and <i>Cochliobolus heterostrophus</i>. 
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<h3>References</h3>
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1. Weld, R. J., Plummer, K. M., Carpenter, M. A., & Ridgeway, H. J. (n.d.). <i>Appraoches to functional genomics in filamentous fungi</i>. N.p.: Nature.
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2. Turgeon, G. B., Condon, B., Liu, J., & Zhang, N. (2010). <i>Protoplast Transformation of Filamentous Fungi</i>  (Vol. 638). N.p.: Molecular and Cell Biology for Fungi.
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3. Feldmann, K. A., & Marks, D. M. (1986). <i> Agrobacterium-mediated transformation of germinating seeds of Arabidopsi thaliana: A non-tissue culture approach.</i> N.p.: Zoecon Research Institute.
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4. Sun, L., Cai, H., Xu, W., Hu, Y., Gao, Y., & Lin, Z. (2001). <i> Efficient Transformation of The Medicinal Mushroom Ganoderma Lucidum</i> (19th ed.). N.p.: Plant Molecular Biology Reporter.
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Latest revision as of 03:04, 29 October 2013

Cornell University Genetically Engineered Machines

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 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.

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.

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.

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