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Team:Cornell/project/wetlab/protoplasting
From 2013.igem.org
Protoplasting
Background
Transformation of plant and fungal cells is difficult due to their cell walls that block passage of foreign DNA. Protoplasting is the method by which the cells walls of plant and fungal cells are digested to produce cells without cell walls, called protoplasts. The protoplasts can then be isolated from canidia by filtering through 20um nylon filter. Through additional methods, such as electroporation and PEG transformation, DNA can be uptaken by the protoplasts and then regrown into cells containing specific genes [1].
Photo: Gillian Turgeon, Cornell University.
Methods
Usually Ganoderma lucidum is protoplasted using lywallzyme and novozyme. However, these enzymes are exclusively available in China. Due to this, protoplasting was attempted with driselease and glucanex instead. After several attempts, we found that the enzymes are unable to digest the cell wall without also killing the cell. In addition, the Ganoderma mycelium was hard to pellet when centrifuging and made the protoplasting procedure difficult. Thus, Cochliobolus heterostrophus was used instead. Protoplasting was successful using driselease and glucanex and then transformed via PEG solution [2].References
1. Sun L, Cai H, Xu W, Hu Y, Gao Y, Lin Z (2001). Efficient Transformation of the Medicinal Mushroom Ganoderma lucidum. Plant Molecular Biology Reporter, 19, 383a-383j.2. Amir Sharon (ed.), Molecular and Cell Biology Methods for Fungi, Methods in Molecular Biology, vol. 638. Turgeon BG, Condon B, Liu J, Zhang N (2010). Protoplast Transformation of Filamentous Fungi.
