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From 2010.igem.org

Project Description

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.

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

Sponsers

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