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From 2010.igem.org

Abstract

Saccharomyces cerevisiae is a model unicellular eukaryotic chassis; however when compared with Escherichia coli the available synthetic biology tools are lacking. To remedy this problem the 2010 Washington University iGEM team has introduced a synthetic alternative splicing tool, as well as designed and produced new BioBricks parts to ease transformation of synthetic constructs into S. cerevisiae. A mutually exclusive exon splicing system was formulated in which Sex-lethal interacts with the native splicing machinery to affect splice site choice. Two vectors have been designed to facilitate simple bacterial BioBrick manipulation and subsequent chromosomal integration into the yeast genome. A yeast positive selection marker BioBrick has been produced for the first time. Chromosomal integration with positive selection will stabilize and streamline BioBrick transformations into S. cerevisiae. A synthetic splicing assembly will allow for new synthetic biology techniques such as isoform engineering of proteins or combinatorial logic.

Yeast: A Simpler Transformation

S. cerevisiae has long served as the model organism of the ekaryotic domain, however the biobrick registry is lacking in terms of the diversity of parts necessary to provide comprehensive support when working with this organism. In order to rectify this problem, the 2010 iGEM team is desiging a set of biobricks that will help simplify the transformation process in S. cerevisiae. Drug resistant genes will be submitted as biobricks allowing for positive selection to be preformed on wildtype yeast strains instead of the more basic supplementation of auxotrophic strains. Furthermore, new plasmid backbrones are to be designed which will allow DNA manipulation to be conducted in E. coli and then direct chromosomal intergration into S. cerevisiae.

Splicing: The New Alternative

The 2010 Washington University iGEM team is designing a synthetic alternative splicing system in Saccharomyces cerevisiae in order to create a new tool synthetic biologists can use in their scientific endeavors. A single definitive locus and only one other potential locus within the S. cerevisiae genome have shown alternative splicing capabilities (Juneau, 2009). This lack of complex splicing activity within S. cerevisiae limits how synthetic biologists utilize splicing in their projects. The 2010 WashU iGEM team strives to overcome this issue by expressing Sex-Lethal (SxL), a Drosophila Melanogaster splicing regulatory gene, in S. cerevisiae and attempting to use it to control alternative splicing events.

The designed construct employs two 3’ splice sites to select for cyan or yellow fluorescent proteins. By altering the presence of SxL within the cell, the preference between the proximal and distal 3' splice sites can be modulated. This results in varying ratios of CFP and YFP, allowing us to show the creation of a synthetically designed alternative splicing mechanism in S. cerevisiae.

The alternative splicing machine can be applied to isoform engineering, showing the unique benefits of this mechanism. A more complex construct is designed in which the 5' end of a flourscent protein is always included in mRNA. However, different 3' ends are alternatively spliced on, creating different isoforms of a protein. This construct simplistically mirrors the much more complex examples of alternative splicing in nature, as in the avian cochlea. Avian cochlear cells alternatively splice as many as 576 different isoforms of the same gene, helping to create a gradient that is necessary to hear a wide spectrum of sound (Black, 1998). Another possible advantage to alternative splicing is that it allows a combinatorial approach to problem solving, like the one used by the 2006 Davidson iGEM team. Instead of using recombinases to modify DNA, splicing, which only affects RNA transcripts, can be used to leave no lasting change in the cell’s genetic code.

Acknowledgements

Sigma Aldrich has generously donated the reagents used during the course of our experiment.

We would like to thank Dean Quatrano and the Washington University in St. Louis Department of Biomedical Engineering for there assistance and funds throughout the entire summer.

We would also like to thank the Washington University in St. Louis Department of Biology and Carol Kohring for their Lab space, equipment and assistance in the Lab.

The Summer Undergraduate Research Fellowship (SURF), Washington University Career Center, Mckelvey Scholarship Program and the Yinjie Lab for providing stipends.

The assistance of the Cohen Lab, Dantas Land, Jez Lab and Yinjie Lab.

Contact Us

If you have any questions or advice we would love to hear from you. The Washington University iGEM team may be reached at WashU.iGEM@gmail.com

Works Cited

Juneau et. al. 2009, Alternative splicing of PTC7 in Saccharomyces cerevisiae determines protein localization, Genetics, v.183, 185-195

Black 1998, Splicing in the inner ear: a familiar tune, but what are the instruments?, Neuron, v. 20, 165-168