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Team:UNIK Copenhagen/Tripartite split GFP
From 2014.igem.org
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<area shape="rect" coords="50,2,92,77" title="Signal peptide" onclick="sigpepFunction();"> | <area shape="rect" coords="50,2,92,77" title="Signal peptide" onclick="sigpepFunction();"> | ||
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<area shape="rect" coords="420,2,495,77" title="Split-GFP 11" onclick="gfp11Function();"> | <area shape="rect" coords="420,2,495,77" title="Split-GFP 11" onclick="gfp11Function();"> | ||
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<area shape="rect" coords="50,2,92,77" title="Signal peptide" onclick="sigpepFunction();"> | <area shape="rect" coords="50,2,92,77" title="Signal peptide" onclick="sigpepFunction();"> | ||
<area shape="rect" coords="94,2,345,77" title="GFP 1-9" onclick="gfp19Function();"> | <area shape="rect" coords="94,2,345,77" title="GFP 1-9" onclick="gfp19Function();"> | ||
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<area shape="rect" coords="50,2,301,77" title="Tobacco Mosaic Virus coating protein" onclick="TMVFunction();"> | <area shape="rect" coords="50,2,301,77" title="Tobacco Mosaic Virus coating protein" onclick="TMVFunction();"> | ||
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function sigpepFunction() { | function sigpepFunction() { | ||
| - | document.getElementById("about_gene").innerHTML="<p>This sequence codes for a signal peptide that is a 19-20 amino acid region in the N-terminal end of proteins. Once translated, | + | document.getElementById("about_gene").innerHTML="<p>This sequence codes for a signal peptide that is a 19-20 amino acid region in the N-terminal end of proteins. Once translated, the signal peptide will bind to a transporter that moves the ribosome to the ER membrane and ensures translation across and into the ER. From here the signal peptide is cleaved off and the rest of the protein can be secreted using the Golgi apparatus.<br><br>The sequence of our signal peptide was taken from the yeast <i>suc2</i> gene, a gene that encodes a constitutively secreted sucrose invertase.</p>"; |
} | } | ||
Revision as of 17:49, 24 August 2014
TRIPARTITE SPLIT GFP
In our split-GFP project we utilize tripartite split GFP (green flourescent protein) fused to FAB (fragment antigen-binding) fragments. The GFP has been split into fragments containing β-strand 1-9, β-strand 10 or β-strand 11. When two FAB fragments with GFP β-strand 10 and 11 bind to the same antigen, they will come close together and fuse with any passing GFP fragments containing β-strand 1-9 with a high affinity. This system could in theory be applied to any molecule or protein containing multiple close-proximity binding sites with known antibodies. 
The capsid proteins of viruses are repetitive structures assembled from a large amount of monomeric units. Therefore antibodies targeting these monomeric units should be able to bind in a large quantity in close proximity.
To achieve this system we found a suitable antigen in the Tobacco Mosaic Virus (TMV), a plant pathogen, and an associated compatible antibody. In our project we construct FAB fragments from this antibody fused with a GFP β-strand 10 or 11 using a flexible linker. By transforming this construct together with a preceding signal peptide, into one line of yeast cells, and the remaining β-strand 1-9 GFP fragment with a preceding signal peptide into another line to avoid GFP fusing within the cells, a mix of these two lines will secrete both types of FAB fragments and the free split GFP 1-9 into their media. When a sample is added to this media, an increase in fluorescence will be indicative of the presence of TMV capsid protein.
Once a yeast strain with a FAB fragment compatible to a desired pathogen is established, production costs of the system should be very low. And due to the low-tech of the finished product, we imagine being able to ship out bags containing dry-yeast and media powder for easy diagnostic field tests in any remote part of the world, with only water, a sample of interest and a UV light being needed.
GENE CONSTRUCTS
Touch the lego bricks to see what sequences the gene consist of and click on the sequences to read more about their function. Note that the information box will be shown under the pictures.
Gene construct 1: HeavyChain-GFP10
Gene construct 2: HeavyChain-GFP11
Gene construct 3: LightChain
Gene construct 4: GFP1-9
Gene construct 5: Antigen
