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Team:WashU/Project Outline
From 2010.igem.org
Splicing, the New Alternative
The 2010 WashU iGEM team is attempting to create and modulate a synthetic splicing mechanism in S. cerevisiae. This will add a valuable new tool into the synthetic biologist's tool-belt that be utilized in many different applications.
Constructs
Three different genetic constructs are being made and tested by this years iGEM team. Each one builds upon the previous construct in our effort to develop and showcase alternative splicing as a synthetic biology tool.
Construct 1
Construct 1 seeks to demonstrate that splicing of the designed intron occurs in S. cerevisiae. To accomplish this it contains two open reading frames, one which codes for CFP and one which codes for YFP. The CFP open reading frame is contained within an intron, causing it to be spliced out. In eukaryotes only the first open reading frame is translated, so in the non-spliced mRNA CFP will be translated and YFP will not be expressed. However in the spliced transcript CFP will be spliced out and YFP will be expressed. Observation of YFP will demonstrate that splicing of the designed intron has occurred within the cell. Observation of CFP will demonstrate that splicing of the designed intron is not 100% effective.
Construct 1 contains the following biobrick parts.
- The Cnstitutive Yeast ADH1 Promoter. [http://partsregistry.org/wiki/index.php?title=Part:BBa_J63005 BBa_J63005]
- The Designed Yeast Kozak Sequence. [http://partsregistry.org/wiki/index.php?title=Part:BBa_J63003 Part:BBa_J63003]
- Engineered Cyan Fluorescent Protein derived from A. victoria GFP. [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0020 BBa_E0020]
- Enhanced Yellow Fluorescent Protein derived from A. victoria GFP. [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0030 BBa_E0030]
