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Team:uOttawa CA/Project
From 2012.igem.org
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Comprehensive Approach to Universal Network Design
The goal of the 2012 uOttawa iGEM team was to continue to work off of the strengths of our 2011 project, which was the construction of gene networks. This year we showcase the benefits of using yeast as a model organism for network construction, characterization and expression. Saccharomyces cerevisiae is a eukaryotic organism that has many physiological properties which makes it superior to E. coli for synthetic biology. The ability to exist in both haploid and diploid states can be used to build networks through mating protocols and its repair mechanisms can be used for a robust assembly of gene networks in vivo.
Network Construction
The first task of building a gene network is to synthesize the sequences that are required for the network. Last year, the uOttawa project showed how PCR could be used to put together many gene dimers (ex. A-B, B-C, C-D) as long as there were homologous regions that could prime the reaction. [Use an * as a hyperlink to last years page] This method worked very well but we continued to improve our protocols to increase efficiency and reduce the amount of time required to construct networks. We adopted the DNA assembler method [1], and optimized it for our purposes. This protocol takes advantage of yeast repair mechanisms and uses homologous recombination to put together networks. By building gene fragments with homologous regions and co-transforming them in yeast we reduced the amount of PCR required to build gene networks. Dimers of gene fragments can be built using traditional BioBrick assembly methods or fusion PCR and co-transformed as per the protocol supplied on our results page.
Shuttle Vector
By building an E.coli/S.cerevisiae shuttle vector we can take advantage of the high reproductive rate of E.coli and the gene synthesis capabilities of S.cerevisiae. Networks will be built via homologous recombination in yeast and replicated in bacteria. Traditional drug selection will be supplemented with colour selection to increase the accuracy of the transformations.
Combinatorial Mating and Gene Regulatory Systems
An advantage of using yeast as a model organism is its ability to exist in both haploid and diploid states. The uOttawa team plans to utilize this ability to combinatorially mate haploid strains to build gene networks. A haploid strain that acts as reporter for gene regulatory proteins can be used as a "testing" strain and mated with other haploid "sample" strains to quickly and efficiently test gene regulatory functions.
Shuttle Vector
By building an E.coli/S.cerevisiae shuttle vector we can take advantage of the high reproductive rate of E.coli and the gene synthesis capabilities of S.cerevisiae. Networks will be built via homologous recombination in yeast and replicated in bacteria. Traditional drug selection will be supplemented with colour selection to increase the accuracy of the transformations.
