Team:UNIK Copenhagen/Tripartite split GFP

From 2014.igem.org

(Difference between revisions)
Line 45: Line 45:
</div>
</div>
<div class="description">
<div class="description">
-
<p align="justify">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. <br> <br>
+
<p align="justify">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. <br><br>
-
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.<br><br>
+
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.<br><br>
-
References:<br>
+
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.
-
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</p><br><br>
+
</p>
-
<p align="justify">We are using homolog recombination to transform the yeast strains.</p><br><br>
 
-
<p align="justify">Gene 1:</p>
 
-
<img src="https://static.igem.org/mediawiki/2014/b/bf/Team_UNIK_Copenhagen_Split_GFP_gene_1.PNG"  usemap="#MapGENE1" border="0">
 
<p align="justify">Touch the lego bricks to see what sequences the gene consist of and click on the sequences to read more about their function.</p>
<p align="justify">Touch the lego bricks to see what sequences the gene consist of and click on the sequences to read more about their function.</p>
 +
 +
<p align="justify">Gene 1 (test):</p>
 +
<img src="https://static.igem.org/mediawiki/2014/b/bf/Team_UNIK_Copenhagen_Split_GFP_gene_1.PNG"  usemap="#MapGENE1" border="0">

Revision as of 19:24, 7 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.

Touch the lego bricks to see what sequences the gene consist of and click on the sequences to read more about their function.

Gene 1 (test):















































Team UNIK Copenhagen
e-mail: igem.cph14@gmail.com



Retrieved from "http://2014.igem.org/Team:UNIK_Copenhagen/Tripartite_split_GFP"