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Team:Cornell/project/wetlab/fungal toolkit/homologous constructs
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Eukaryotic cells can incorporate foreign DNA through random insertion and homologous recombination. In random insertion, foreign DNA is randomly integrated into the genome through a process that is currently not well understood. Homologous recombination is the process by which DNA is exchanged between a genome and a construct in regions of homology. Thus, we can place a gene of interest 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. | Eukaryotic cells can incorporate foreign DNA through random insertion and homologous recombination. In random insertion, foreign DNA is randomly integrated into the genome through a process that is currently not well understood. Homologous recombination is the process by which DNA is exchanged between a genome and a construct in regions of homology. Thus, we can place a gene of interest 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. | ||
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Revision as of 02:36, 28 September 2013
Homologous Constructs
Background
Eukaryotic cells can incorporate foreign DNA through random insertion and homologous recombination. In random insertion, foreign DNA is randomly integrated into the genome through a process that is currently not well understood. Homologous recombination is the process by which DNA is exchanged between a genome and a construct in regions of homology. Thus, we can place a gene of interest 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 using a three part gibson assembly. The three 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 [1].
