Background
Two important methods whereby eukaryotic cells incorporate foreign DNA is random insertion and homologous recombination. In random insertion, foreign DNA is randomly integrated into the genome. The biology of this process is not exactly known. Homologous recombination is the process by which DNA is exchanged between a genome and a construct in areas of homology. Thus, a gene of interest can be placed between known regions of homology in order to increase its chances of being incorporated into the genome. This process has been noted to be especially effective in transforming plant and fungal species.
Method
Constructs were made containing selected genes flanked by regions homologous to the Ganoderma lucidum gpd promoter. Thus, the construct would be compatible with homologous recombination. The construct was made with a 3 part gibson assembly. The 3 parts were all made in separate biobricks containing the left and right homology regions and the selected genes. The genes inserted into the homology flanking regions include prtpc promoter, gpd promoter, t7 promoter, and the resistance genes hph and nptII . The constructs were then inserted into the respective fungus strain using protoplasting and PEG transformation methods.
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