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Team:UNIK Copenhagen/Tripartite split GFP
From 2014.igem.org
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</div> | </div> | ||
<div class="description"> | <div class="description"> | ||
| - | <p><br><b>Touch</b> the lego bricks to see what sequences the gene consist of and <b>click</b> on the sequences to read more about their function.</p> | + | <p><br><b>Touch</b> the lego bricks to see what sequences the gene consist of and <b>click</b> on the sequences to read more about their function. Note that the information box will be shown under the pictures.</p> |
<p>Gene construct 1: HeavyChain-GFP10</p> | <p>Gene construct 1: HeavyChain-GFP10</p> | ||
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function can1Function() { | function can1Function() { | ||
| - | document.getElementById("about_gene").innerHTML="<p>40 bp | + | document.getElementById("about_gene").innerHTML="<p><i>can1</i> is a 40 bp sequence identical to sequences flanking the ORF in the CAN1 gene of Saccharomyces cerevisiae. These allow for homologous recombination of our genes directly into the yeast genome, replacing the existing protein product, a transmembrane arginine transporter, while still using the existing promoter region. This transporter also allows the uptake of the toxic compound Canavanine, allowing us to select for transformants as they will lack the transporter.</p>"; |
} | } | ||
function gfp10Function() { | function gfp10Function() { | ||
| - | document.getElementById("about_gene").innerHTML="<p> | + | document.getElementById("about_gene").innerHTML="<p>This sequence codes for 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> | + | document.getElementById("about_gene").innerHTML="<p>This sequence codes for 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>"; |
} | } | ||
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function linkFunction() { | function linkFunction() { | ||
| - | document.getElementById("about_gene").innerHTML="<p> | + | document.getElementById("about_gene").innerHTML="<p>This sequence is a repeating Gly-Gly-Gly-Gly-Ser peptide containing 13 repetitions, allowing it to span one half of the distance between two binding sites on the Tobacco Mosaic Virus Capsid Protein. This allows the split GFP peptides of two adjacent bound FAB fragments to reach each other. The (Gly4Ser)n peptide is a commonly linker with high flexibility and water solubility.</p>"; |
} | } | ||
function TMVFunction() { | function TMVFunction() { | ||
| - | document.getElementById("about_gene").innerHTML="<p> | + | document.getElementById("about_gene").innerHTML="<p>This sequence codes for the coating protein the of the Tobacco Mosaic Virus, this serves as our antigen. A single protein is only 158 amino acids long, but several thousand such proteins will self-assemble into the long spiraling Capsid protein with 16.3 proteins per helical turn. With antibody binding sites on the outside of the spiral and a diameter of 18 nm, amounting to a 3½ nm distance between each site, this should supply an ample amount of binding sites for our FAB fragments.<br><br>Sequence obtained from UniProt</p>"; |
} | } | ||
Revision as of 16:21, 16 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. 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
