Team:UNIK Copenhagen/Safety

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Revision as of 13:27, 14 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 construct 1: HeavyChain-GFP10

Gene construct 2: HeavyChain-GFP11

Gene construct 3: LightChain

Gene construct 4: GFP1-9

Gene construct 5: Antigen

@@ Line 161: / Line 161: @@
 }
-function GFP10Function() {
+function gfp10Function() {
      document.getElementById("about_gene").innerHTML="<p>The 10’th β-strand of Green Fluorescent Protein (GFP), when combined with strand 1-9 and 11 will fuse to functional fluorescing GFP.<br><br>Sequence obtained from paper describing tripartite split-GFP</p>";
 }
-function GFP11Function() {
+function gfp11Function() {
      document.getElementById("about_gene").innerHTML="<p>The 11’th β-strand of Green Fluorescent Protein (GFP), when combined with strand 1-9 and 10 will fuse to functional fluorescing GFP.<br><br>Sequence obtained from paper describing tripartite split-GFP</p>";
 }
-function GFP19Function() {
+function gfp19Function() {
      document.getElementById("about_gene").innerHTML="<p>Green Fluorescent Protein (GFP) lacking the 10’th and 11’th β-strands, when recombined with strand 10 and 11 will fuse to functional fluorescing GFP.<br><br>Sequence obtained from paper describing tripartite split-GFP</p>";
 }