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Team:Cornell/project/wetlab/fungal toolkit/fungal transformation
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| - | Fluorescence microscopy showed evidence of GFP expression in fungal mycelium. | + | Fluorescence microscopy showed evidence of GFP expression in fungal mycelium transformed with a construct containing GFP under the control of the trpc promoter flanked by homology regions of <i>Cochliobolus heterostrophus</i> GPD. Wild type strains showed no signs of innate fluoresence.<br> |
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<h3>Agrobacterium Mediated Transformation</h3> | <h3>Agrobacterium Mediated Transformation</h3> | ||
<h5>Background</h5> | <h5>Background</h5> | ||
Revision as of 02:50, 29 October 2013
Fungal Transformation
PEG Mediated Transformation
Background
Methods
Transformants
Agrobacterium Mediated Transformation
Background
Wide varieties of plant and fungal cells can be easily and efficiently transformed by agrobacterium-mediated transformation. In this process, plant or fungal tissue is co-cultured with Agrobacterium tumefaciens, an organism that is able to transfer part of its DNA to plants and fungi, known as T-DNA. Genetic constructs can be inserted into these T-DNA vectors and then transformed into A. tumefaciens, allowing it to insert this construct into a plant or fungal genome [1].
Methods
In order to transform our genetic constructs into Ganoderma lucidum, we first inserted them into pOSCAR, a T-DNA vector that contains regions that A. tumefaciens can insert into fungal cells. Following transformation of A. tumefaciens with our constructs in the pOSCAR vector, we co-cultured the cells with G. lucidum in induction media that provides optimal conditions for T-DNA insertion for a period of 2 days. G. lucidum was then placed on a selective plate to isolate transformants [2].References
1. Michielse, Caroline B., Paul J. J. Hooykaas, Cees A. M. J. J. Hondel, and Arthur F. J. Ram. "Agrobacterium-mediated Transformation as a Tool for Functional Genomics in Fungi." Current Genetics 48.1 (2005): 1-17.2. Paz, Zahi, María D. García-Pedrajas, David L. Andrews, Steven J. Klosterman, Lourdes Baeza-Montañez, and Scott E. Gold. "One Step Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), an Efficient and Robust Tool for ATMT Based Gene Deletion Construction in Fungi." Fungal Genetics and Biology (2011): 677-684.
