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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.
Once the gene constructs were assembled and transformed into yeast we were able to take advantage of yeast mating protocols to build larger networks. This allows for the rapid integration of constructs to build large networks without the time commitment of multiple transformation procedures. For example, simultaneously making haploid strains of two constructs each, can build a gene network composed of four constructs once mated together. Furthermore, if one gene module is required in multiple networks it can be built into one mating type haploid (a or α) and mated with many different gene modules that have been integrated into the opposite mating type.
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.
Characterization of Network Components
A large roadblock on the construction of large gene networks has been the predictability of the components. In order to construct a network with desired function, the parts need to first be characterized and compared to a standard reference strain. The reference strain acts to set a basal expression rate for testing strains and other inducible systems to compare their expression levels to.
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.
