Team:UNIK Copenhagen/Tripartite split GFP

From 2014.igem.org

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<div class="the_content">
<div class="subject">
<div class="subject">
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<h4>TRIPARTITE SPLIT GFP</h4>
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<h3>TRIPARTITE SPLIT GFP</h3>
</div>
</div>
<div class="description">
<div class="description">
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<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>
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<p>In our split-GFP project we utilize tripartite split <abbr title="Green flourescent protein">GFP</abbr> fused to <abbr title="Fragment antigen binding">FAB</abbr> fragments. The GFP has been split into fragments containing β-strand 1-9, β-strand 10 or β-strand 11. When two FAB fragments with GFP β-strand 10 and 11 bind to the same antigen, they will come 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. <img src="https://static.igem.org/mediawiki/2014/4/45/Team_UNIK_Copenhagen_Split_GFP_illustration.PNG" class="right"> 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>
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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>
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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>
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References:<br>
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<img src="https://static.igem.org/mediawiki/2014/4/42/Team_UNIK_Copenhagen_Split_GFP_illustration2.PNG">
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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>
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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, a sample of interest and a UV light being needed.<br>
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</p>
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</div>
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<p align="justify">We are using homolog recombination to transform the yeast strains.</p><br><br>
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<div class="subject">
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<p align="justify">Gene 1:</p>
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<h3>GENE CONSTRUCTS</h3>
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<img src="https://static.igem.org/mediawiki/2014/b/bf/Team_UNIK_Copenhagen_Split_GFP_gene_1.PNG"  usemap="#MapGENE1" border="0">
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</div>
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<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>
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<div class="description">
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<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>
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<p>Gene construct 1: HeavyChain-GFP10</p>
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<img src="https://static.igem.org/mediawiki/2014/4/46/Team_UNIK_Copenhagen_Split_GFP_construct_1_and_2.png"  usemap="#MapGENE1" border="0">
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<br>
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<p>Gene construct 2: HeavyChain-GFP11</p>
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<img src="https://static.igem.org/mediawiki/2014/4/46/Team_UNIK_Copenhagen_Split_GFP_construct_1_and_2.png"  usemap="#MapGENE2" border="0">
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<br>
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<p>Gene construct 3: LightChain</p>
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<img src="https://static.igem.org/mediawiki/2014/6/64/Team_UNIK_Copenhagen_Split_GFP_construct_3.png"  usemap="#MapGENE3" border="0">
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<br>
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<p>Gene construct 4: GFP1-9</p>
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<img src="https://static.igem.org/mediawiki/2014/0/0c/Team_UNIK_Copenhagen_Split_GFP_construct_4.png"  usemap="#MapGENE4" border="0">
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<br>
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<p>Gene construct 5: Antigen</p>
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<img src="https://static.igem.org/mediawiki/2014/8/8c/Team_UNIK_Copenhagen_Split_GFP_construct_5.png"  usemap="#MapGENE5" border="0">
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<p><br></p>
<map name="MapGENE1">
<map name="MapGENE1">
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   <area shape="rect" coords="7,9,50,55" title="Flanking side: can1" type="button" onclick="can1Function();">
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   <area shape="rect" coords="4,2,55,51" title="Flanking side: CAN1" type="button" onclick="can1Function();">
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   <area shape="rect" coords="57,9,118,55" title="suc2" onclick="suc2Function();">
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   <area shape="rect" coords="57,2,141,51" title="Signal peptide" onclick="sigpepFunction();">
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   <area shape="rect" coords="124,9,308,55" title="FAB" onclick="fabFunction();">
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   <area shape="rect" coords="142,2,252,51" title="Variable domain of the heavy chain" onclick="hcvFunction();">
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   <area shape="rect" coords="313,9,403,55" title="GFP 1-9" onclick="gfpFunction();">
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   <area shape="rect" coords="253,2,362,51" title="Conserved domain of the heavy chain" onclick="hccFunction();">
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    <area shape="rect" coords="408,9,451,55" title="Flanking side: can1" onclick="can1Function();">
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  <area shape="rect" coords="264,2,419,51" title="Linker" onclick="linkFunction();">
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  <area shape="rect" coords="420,2,503,51" title="Split-GFP 10" onclick="gfp10Function();">
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  <area shape="rect" coords="504,2,560,51" title="Flanking side: CAN1" onclick="can1Function();">
</map>
</map>
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<map name="MapGENE2">
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  <area shape="rect" coords="4,2,55,51" title="Flanking side: can1" type="button" onclick="can1Function();">
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  <area shape="rect" coords="57,2,141,51" title="Signal peptide" onclick="sigpepFunction();">
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  <area shape="rect" coords="142,2,252,51" title="Variable domain of the heavy chain" onclick="hcvFunction();">
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  <area shape="rect" coords="253,2,362,51" title="Conserved domain of the heavy chain" onclick="hccFunction();">
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  <area shape="rect" coords="264,2,419,51" title="Linker" onclick="linkFunction();">
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  <area shape="rect" coords="420,2,503,51" title="Split-GFP 11" onclick="gfp11Function();">
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  <area shape="rect" coords="504,2,560,51" title="Flanking side: can1" onclick="can1Function();">
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</map>
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<table class="sequence_description" id="about_gene"><p></p>
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<map name="MapGENE3">
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</table></div>
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  <area shape="rect" coords="4,2,55,51" title="Flanking side: ura3" type="button" onclick="ura3Function();">
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  <area shape="rect" coords="57,2,141,51" title="Signal peptide" onclick="sigpepFunction();">
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  <area shape="rect" coords="142,2,252,51" title="Variable domain of the light chain" onclick="lcvFunction();">
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  <area shape="rect" coords="253,2,362,51" title="Conserved domain of the light chain" onclick="lccFunction();">
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  <area shape="rect" coords="264,2,421,51" title="Flanking side: ura3" onclick="ura3Function();">
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</map>
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<map name="MapGENE4">
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  <area shape="rect" coords="2,2,57,51" title="Flanking side: can1" type="button" onclick="can1Function();">
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  <area shape="rect" coords="58,2,137,51" title="Signal peptide" onclick="sigpepFunction();">
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  <area shape="rect" coords="138,2,247,51" title="GFP 1-9" onclick="gfp19Function();">
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  <area shape="rect" coords="248,2,300,51" title="Flanking side: can1" onclick="can1Function();">
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</map>
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<map name="MapGENE5">
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  <area shape="rect" coords="4,2,56,51" title="Flanking side: can1" type="button" onclick="can1Function();">
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  <area shape="rect" coords="57,2,162,51" title="Tobacco Mosaic Virus coating protein" onclick="TMVFunction();">
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  <area shape="rect" coords="163,2,215,51" title="Flanking side: can1" onclick="can1Function();">
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</map>
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<table class="sequence_description" id="about_gene"></table></div>
<script>
<script>
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function ura3Function() {
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    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>";
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}
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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, the signal peptide will bind to a 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 the rest of the protein can be secreted using the Golgi apparatus.<br><br>The sequence of our signal peptide was taken from the yeast <i>suc2</i> gene, a gene that encodes a constitutively secreted sucrose invertase.</p>";
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}
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function lcvFunction() {
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    document.getElementById("about_gene").innerHTML="<p>This sequence encodes 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.<br><br>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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}
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function lccFunction() {
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    document.getElementById("about_gene").innerHTML="<p>This sequence encodes 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.<br><br>The sequence of our light chain conserved domain was obtained from UniProt.</p>";
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}
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function hcvFunction() {
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    document.getElementById("about_gene").innerHTML="<p>This sequence encode a heavy chain variable domain. When structurally adjacent to a light chain variable domain, these sequences encode the antigen binding part 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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}
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function hccFunction() {
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    document.getElementById("about_gene").innerHTML="<p>This sequence encode a light chain conserved domain. Fused with the heavy chain variable domain, these two domains makes up the entire heavy chain of our FAB fragment. Containing an exposed cysteine, this will form a disulfide bridge to a similar exposed cysteine on the Light Chain Conserved domain. Once bound together, the Heavy and Light Chains will form the finished FAB fragment.<br><br>The sequence of our Heavy Chain Conserved domain is the CH1 domain of a full Heavy Chain obtained from UniProt.</p>";
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}
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function can1Function() {
function can1Function() {
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     document.getElementById("about_gene").innerHTML="can1 codes for a transporter that transport arginin and canavanin into the cell";
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     document.getElementById("about_gene").innerHTML="<p><i>can1</i> is a 40 bp sequence identical to sequences flanking the ORF in the <i>can1</i> 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 and thus can be grown on a media containing Canavanine.</p>";
}
}
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function suc2Function() {
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function gfp10Function() {
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     document.getElementById("about_gene").innerHTML="suc2 codes for a signal sequence that localizes the translation to ER";
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     document.getElementById("about_gene").innerHTML="<p>This sequence codes for the 10<sup>th</sup> β-strand of Green Fluorescent Protein (GFP). When it is combined with strand 1-9 and 11 then they become a functional fluorescing GFP.<br><br>The GFP sequences are obtained from a paper describing tripartite split-GFP</p>";
}
}
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function fabFunction() {
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function gfp11Function() {
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     document.getElementById("about_gene").innerHTML="this seguence codes for the FAB fragment";
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     document.getElementById("about_gene").innerHTML="<p>This sequence codes for the 11<sup>th</sup> β-strand of Green Fluorescent Protein (GFP). When it is combined with strand 1-9 and 10 then they will become a functional fluorescing GFP.<br><br>The GFP sequences are obtained from a paper describing tripartite split-GFP</p>";
}
}
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function gfpFunction() {
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function gfp19Function() {
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     document.getElementById("about_gene").innerHTML="this gene codes for one of the GFP fragments";
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     document.getElementById("about_gene").innerHTML="<p>GFP lacking the 10<sup>th</sup> and 11<sup>th</sup> β-strands. When this GFP fragment is recombined with strand 10 and 11, they will become functional fluorescing GFP.<br><br>The GFP sequences are obtained from a paper describing tripartite split-GFP.</p>";
}
}
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</script>
 
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function linkFunction() {
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    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 (Gly4-Ser)n peptide is a commonly linker with high flexibility and water solubility.</p>";
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}
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function TMVFunction() {
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    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>This sequence is obtained from UniProt</p>";
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}
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<p>Team UNIK Copenhagen <br> <b>e-mail:</b> igem.cph14@gmail.com<p><br>
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Latest revision as of 18:23, 11 September 2014




TRIPARTITE SPLIT GFP

In our split-GFP project we utilize tripartite split GFP fused to FAB fragments. The GFP has been split into fragments containing β-strand 1-9, β-strand 10 or β-strand 11. When two FAB fragments with GFP β-strand 10 and 11 bind to the same antigen, they will come 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, a 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



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