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Team:UNIK Copenhagen/Tripartite split GFP
From 2014.igem.org
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Revision as of 12:41, 12 August 2014
TRIPARTITE SPLIT GFP
In our split-GFP project we utilize tripartite split GFP fused to FAB (fragment antigen-binding) fragments so that when two FAB fragments with GFP β-strand 10 and 11 bind to the same antigen, both β-strands will always be 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, 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.
Gene constuct 3:
The other gene constructs will come soon!
