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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>In our split-GFP project we utilize tripartite split | + | <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> |
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> | 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> | ||
<img src="https://static.igem.org/mediawiki/2014/4/42/Team_UNIK_Copenhagen_Split_GFP_illustration2.PNG"> | <img src="https://static.igem.org/mediawiki/2014/4/42/Team_UNIK_Copenhagen_Split_GFP_illustration2.PNG"> | ||
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<p>Gene construct 1: HeavyChain-GFP10</p> | <p>Gene construct 1: HeavyChain-GFP10</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2014/ | + | <img src="https://static.igem.org/mediawiki/2014/4/46/Team_UNIK_Copenhagen_Split_GFP_construct_1_and_2.png" usemap="#MapGENE1" border="0"> |
| - | + | <br> | |
<p>Gene construct 2: HeavyChain-GFP11</p> | <p>Gene construct 2: HeavyChain-GFP11</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2014/ | + | <img src="https://static.igem.org/mediawiki/2014/4/46/Team_UNIK_Copenhagen_Split_GFP_construct_1_and_2.png" usemap="#MapGENE2" border="0"> |
| - | + | <br> | |
<p>Gene construct 3: LightChain</p> | <p>Gene construct 3: LightChain</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2014/6/ | + | <img src="https://static.igem.org/mediawiki/2014/6/64/Team_UNIK_Copenhagen_Split_GFP_construct_3.png" usemap="#MapGENE3" border="0"> |
| - | + | <br> | |
<p>Gene construct 4: GFP1-9</p> | <p>Gene construct 4: GFP1-9</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2014/ | + | <img src="https://static.igem.org/mediawiki/2014/0/0c/Team_UNIK_Copenhagen_Split_GFP_construct_4.png" usemap="#MapGENE4" border="0"> |
| - | + | <br> | |
<p>Gene construct 5: Antigen</p> | <p>Gene construct 5: Antigen</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2014/ | + | <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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<map name="MapGENE1"> | <map name="MapGENE1"> | ||
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="4,2,55,51" title="Flanking side: CAN1" type="button" onclick="can1Function();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="57,2,141,51" title="Signal peptide" onclick="sigpepFunction();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="142,2,252,51" title="Variable domain of the heavy chain" onclick="hcvFunction();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="253,2,362,51" title="Conserved domain of the heavy chain" onclick="hccFunction();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="264,2,419,51" title="Linker" onclick="linkFunction();"> |
| - | <area shape="rect" coords="420, | + | <area shape="rect" coords="420,2,503,51" title="Split-GFP 10" onclick="gfp10Function();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="504,2,560,51" title="Flanking side: CAN1" onclick="can1Function();"> |
</map> | </map> | ||
<map name="MapGENE2"> | <map name="MapGENE2"> | ||
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="4,2,55,51" title="Flanking side: can1" type="button" onclick="can1Function();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="57,2,141,51" title="Signal peptide" onclick="sigpepFunction();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="142,2,252,51" title="Variable domain of the heavy chain" onclick="hcvFunction();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="253,2,362,51" title="Conserved domain of the heavy chain" onclick="hccFunction();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="264,2,419,51" title="Linker" onclick="linkFunction();"> |
| - | <area shape="rect" coords="420,2, | + | <area shape="rect" coords="420,2,503,51" title="Split-GFP 11" onclick="gfp11Function();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="504,2,560,51" title="Flanking side: can1" onclick="can1Function();"> |
</map> | </map> | ||
<map name="MapGENE3"> | <map name="MapGENE3"> | ||
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="4,2,55,51" title="Flanking side: ura3" type="button" onclick="ura3Function();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="57,2,141,51" title="Signal peptide" onclick="sigpepFunction();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="142,2,252,51" title="Variable domain of the light chain" onclick="lcvFunction();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="253,2,362,51" title="Conserved domain of the light chain" onclick="lccFunction();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="264,2,421,51" title="Flanking side: ura3" onclick="ura3Function();"> |
</map> | </map> | ||
<map name="MapGENE4"> | <map name="MapGENE4"> | ||
| - | <area shape="rect" coords="2,2, | + | <area shape="rect" coords="2,2,57,51" title="Flanking side: can1" type="button" onclick="can1Function();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="58,2,137,51" title="Signal peptide" onclick="sigpepFunction();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="138,2,247,51" title="GFP 1-9" onclick="gfp19Function();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="248,2,300,51" title="Flanking side: can1" onclick="can1Function();"> |
</map> | </map> | ||
<map name="MapGENE5"> | <map name="MapGENE5"> | ||
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="4,2,56,51" title="Flanking side: can1" type="button" onclick="can1Function();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="57,2,162,51" title="Tobacco Mosaic Virus coating protein" onclick="TMVFunction();"> |
| - | <area shape="rect" coords=" | + | <area shape="rect" coords="163,2,215,51" title="Flanking side: can1" onclick="can1Function();"> |
</map> | </map> | ||
| Line 140: | Line 140: | ||
function sigpepFunction() { | function sigpepFunction() { | ||
| - | 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, | + | 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>"; |
} | } | ||
function lcvFunction() { | function lcvFunction() { | ||
| - | document.getElementById("about_gene").innerHTML="<p>This sequence | + | 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>"; |
} | } | ||
function lccFunction() { | function lccFunction() { | ||
| - | document.getElementById("about_gene").innerHTML="<p>This sequence | + | 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>"; |
} | } | ||
function hcvFunction() { | function hcvFunction() { | ||
| - | document.getElementById("about_gene").innerHTML="<p>When structurally adjacent to a | + | 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>"; |
} | } | ||
function hccFunction() { | function hccFunction() { | ||
| - | document.getElementById("about_gene").innerHTML="<p>Fused with the | + | 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>"; |
} | } | ||
| Line 164: | Line 164: | ||
function gfp10Function() { | function gfp10Function() { | ||
| - | document.getElementById("about_gene").innerHTML="<p>This sequence codes for the | + | 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>"; |
} | } | ||
function gfp11Function() { | function gfp11Function() { | ||
| - | document.getElementById("about_gene").innerHTML="<p>This sequence codes for the | + | 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>"; |
} | } | ||
function gfp19Function() { | function gfp19Function() { | ||
| - | document.getElementById("about_gene").innerHTML="<p> | + | 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>"; |
} | } | ||
function linkFunction() { | function linkFunction() { | ||
| - | 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 ( | + | 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>"; |
} | } | ||
function TMVFunction() { | function TMVFunction() { | ||
| - | 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 | + | 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>"; |
} | } | ||
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
