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Team:UNIK Copenhagen/Tripartite split GFP
From 2014.igem.org
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Revision as of 20:14, 6 August 2014
TRIPARTITE SPLIT GFP
We split GFP into one large and two small fragments that when put in close-proximity to each other will fuse together into a functional GFP. We then link the two small GFP fragments to antibodies on a genetic level and having the third fragment unbound. Antigens with multiple epitopes can then bind those engineered antibodies, bringing the GFP fragments into close proximity. This will lead to the formation of functional GFP in presence of antigens and the large, unbound GFP part. By cloning this system into yeast (Saccharomyces cerevisiae) and making it secrete all three parts, we will arrive at a new single step detection tool where a sample, mixed with water and yeast, will prove the presence of a targeted pathogen by an increase in fluorescence. Once a stable yeast strain is obtained, targeting a specific antigen, simple scale up would allow for mass production and chemical usage minimized. Therefore, this tool should be both cheap and environmentally responsible compared to existing detection methods such as ELISA. The simplicity of this system would allow for dried yeast to be send across the world for easy field-testing.
Our split-GFP approach was inspired by the work of (Cabantous et al., 2013) who used it for protein-protein interactions. Realising that if we replaced the interacting proteins with antibodies with close binding sites, an antigen detector would be possible.
References:
Cabantous, S., Nguyen, H. B., Pedelacq, J.-D., Koraïchi, F., Chaudhary, A., Ganguly, K., … Waldo, G. S. (2013). A new protein-protein interaction sensor based on tripartite split-GFP association. Scientific Reports, 3, 2854. doi:10.1038/srep02854
We are using homolog recombination to transform the yeast strains.
Gene 1:
Touch the lego bricks to see what sequences the gene consist of and click on the sequences to read more about their function.
