Team:UNIK Copenhagen/Tripartite split GFP

From 2014.igem.org

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<script>
<script>
function ura3Function() {
function ura3Function() {
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     document.getElementById("about_gene").innerHTML="<p>The ura3 sequence consist of 40 bp identical to sequences flanking the ORF in the ura3 gene of Saccharomyces cerevisiae. These allow for homologous recombination of our genes directly into the yeast genome, replacing the existing protein product, OCDase, while still using the existing promoter region. OCDase is an enzyme involved in Uracil synthesis, also capable of converting 5-Flourooric Acid into toxic compound 5-Florouracil, causing cell death. This allows us to select for transformants having the ura3 gene replaced by our gene insert.</p>";
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     document.getElementById("about_gene").innerHTML="<p>The ura3 sequence consist of 40 bp identical to sequences flanking the ORF in the ura3 gene of Saccharomyces cerevisiae. </p>";
}
}
function sigpepFunction() {
function sigpepFunction() {
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     document.getElementById("about_gene").innerHTML="<p>This sequence codes for a signal peptide that is a 19-20 amino acid region in the N-terminal end of proteins. Once translated, this will bind to transporter that moves the ribosome to the ER membrane and ensures translation across and into the ER. From here the signal peptide is cleaved off and rest of the protein can be secreted using the Golgi apparatus.
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     document.getElementById("about_gene").innerHTML="<p>This sequence codes for a signal peptide that is a 19-20 amino acid region in the N-terminal end of proteins.</p>";
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The sequence of our signal peptide was taken from the yeast suc2 gene, a gene that encodes a constitutively secreted sucrose invertase.</p>";
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}
}
function lcvFunction() {
function lcvFunction() {
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     document.getElementById("about_gene").innerHTML="<p>This sequence codes for the variable domain of the light chain. When structurally adjacent to a Heavy Chain Variable domain, these sequences encode the antigen binding domain of an Antibody. Our Variable domain sequences originate from a paper describing the variable regions of monoclonal mouse antibodies against Tobacco Mosaic Virus.</p>";
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     document.getElementById("about_gene").innerHTML="<p>This sequence codes for the variable domain of the light chain.</p>";
}
}
function lccFunction() {
function lccFunction() {
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     document.getElementById("about_gene").innerHTML="<p>This sequence codes for the conserved domain of the light chain. Fused with the Light Chain Variable domain, these two domains makes up the entire Light Chain of our FAB fragment. Containing an exposed cysteine, this will form a disulfide bridge to a similar exposed cysteine on the Heavy Chain Conserved domain. Once bound together, the Light and Heavy Chains will form the finished FAB fragment.
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     document.getElementById("about_gene").innerHTML="<p>This sequence codes for the conserved domain of the light chain.</p>";
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The sequence of our Light Chain Conserved domain was obtained from UniProt.</p>";
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}
}

Revision as of 12:50, 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 construct 3:

The other gene constructs will come soon!


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