"
Team:UNIK Copenhagen/Tripartite split GFP
From 2014.igem.org
| Line 91: | Line 91: | ||
<script> | <script> | ||
function ura3Function() { | function ura3Function() { | ||
| - | document.getElementById("about_gene").innerHTML="<p>The ura3 sequence consist of 40 bp identical to sequences flanking the ORF in the <i>ura3</i> 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.<br><br> 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>"; | + | document.getElementById("about_gene").innerHTML="<p>The <i>ura3</i> sequence consist of 40 bp identical to sequences flanking the ORF in the <i>ura3</i> 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.<br><br> 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 <i>ura3</i> gene replaced by our gene insert.</p>"; |
} | } | ||
| Line 99: | Line 99: | ||
function lcvFunction() { | function lcvFunction() { | ||
| - | document.getElementById("about_gene").innerHTML="<p>This sequence codes for the variable domain of the light chain.</p>"; | + | 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>"; |
} | } | ||
Revision as of 13:04, 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!
