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Team:Linkoping Sweden/Project/Biology
From 2014.igem.org
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| - | <p>Our approach is to use an antibody in combination with a fluorescent probe to detect the Ara | + | <p>Our approach is to use an antibody in combination with a fluorescent probe to detect the Ara h1 antigen. This system will have three potential parts: an antibody complex consisting of an polyclonal or monoclonal antibody connected to a fluorescent probe, a default-epitope complex consisting of one or five epitope(s) linked to a fluorescent probe (number of epitopes dependent on antibody class), and the Ara h1 protein. These are potential parts in the sense that the system will always contain the first two parts (antibody complex and the default epitope complex), but will only contain the third part, the Ara h1 protein, if the inserted sample has been contaminated with peanut protein. The biosensor system is to be implemented in a device consisting of a box containing the sensors necessary to detect fluorescence and any eventual changes in fluorescence. This information is then processed by a microprocessor to give a color indication, red or green, correlating to the changes in fluorescence. So how exactly will the system work? Well, there are two possible scenarios for our biosensor system, either the sample does not contain peanut protein (Ara h) (Fig. 1A) or it does (Fig. 1B). </p> |
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| - | <div class="text-panel" style=" | + | <div class="text-panel two-columns"> |
| - | <h1>Scenario: No peanut protein | + | <div style="display: inline-block;"> |
| - | + | <h1>Scenario: No peanut protein contamination</h1> | |
| - | + | <p>The first scenario is default for our biosensor; it contains two interacting parts in the form of an antibody connected to a fluorescent probe (Fluorescein isothiocyanate, or FITC) and epitope(s) bound to a fluorescent probe (Red Fluorescence Protein or RFP). The epitope(s) bind to the antibody, and when a light source is shone on the biosensor, the incoming light is absorbed by FITC and transferred to RFP through Förster Resonance Energy Transfer (FRET) (Fig. 2). The emitted wavelength is detected and then processed by a microprocessor which makes a LED light-panel shine green. Green light thus indicates that the system only contains the antibody and epitope complexe, and has not been contaminated by peanut protein.</p> | |
| - | + | </div> | |
| - | <p>The first scenario is default for our biosensor; it contains two interacting parts in the form of an antibody connected to a fluorescent probe (Fluorescein isothiocyanate, or FITC) and epitope(s) bound to a fluorescent probe (Red Fluorescence Protein or RFP). The epitope(s) | + | |
| - | <div class="image-box" style="width: | + | <div class="image-box" style="width:350px;padding-top:0px;"> |
| - | <a href="# | + | <a href="#fig1a" name="modal"> |
| - | <img src="https://static.igem.org/mediawiki/2014/ | + | <img src="https://static.igem.org/mediawiki/2014/8/87/Linkoping_sweden_MekanismWEBstanding-A.jpg" width="350px" height="auto" title="No peanut protein contamination"></a> |
<div id="boxes"> | <div id="boxes"> | ||
| - | <div id=" | + | <div id="fig1a" class="window" style="width:646px;height:402px"> |
| - | <img src="https://static.igem.org/mediawiki/2014/ | + | <img src="https://static.igem.org/mediawiki/2014/8/87/Linkoping_sweden_MekanismWEBstanding-A.jpg" width="646px" height="402px" title="No peanut protein contamination"> |
| - | <p>Fig | + | <p>Fig 1A. No peanut protein contamination</p> |
</div> | </div> | ||
<div id="mask"></div> | <div id="mask"></div> | ||
</div> | </div> | ||
| - | <p> | + | <p>Figure 1A. No peanut protein contamination. When the cuvette has been loaded with a sample that doesn't contain any peanut, the antibody-antigen complex remains unchanged. (Click to enlarge).</p> |
</div> | </div> | ||
</div> | </div> | ||
| - | <div class="text-panel" style=" | + | <div class="text-panel two-columns"> |
| + | <div style="display: inline-block;"> | ||
<h1>Scenario: Peanut protein contamination</h1> | <h1>Scenario: Peanut protein contamination</h1> | ||
| + | <p>The other possible scenario is that the inserted food sample has been contaminated by peanut and that the system thus contains Ara h1. The peanut protein will bind with a higher affinity to the FITC-antibody complex, thus forcing the epitope-RFP complex out of the picture. Ara h1 bound to the antibody-FITC complex reduces FRET as applied light is absorbed and emitted solely by FITC. This results in a wavelength change which is detected and processed, causing a LED light-panel switch from green to red. Red light thus indicates that the system has been contaminated by peanut and that the food is unsafe to consume. | ||
| + | </p> | ||
| + | </div> | ||
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| + | <div class="image-box" style="width:350px;padding-top:0px;"> | ||
| + | <a href="#fig1b" name="modal"> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/0/07/Linkoping_sweden_MekanismWEBstanding-B.jpg" width="350px" height="auto" title="Peanut protein contamination"></a> | ||
| + | <div id="boxes"> | ||
| + | <div id="fig1b" class="window" style="width:646px;height:372px"> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/0/07/Linkoping_sweden_MekanismWEBstanding-B.jpg" width="646px" height="372px" title="Peanut protein contamination"> | ||
| + | <p>Fig 1B. Peanut protein contamination</p> | ||
| + | </div> | ||
| + | <div id="mask"></div> | ||
| + | </div> | ||
| + | <p>Figure 1B. Peanut protein contamination. When the loaded food-sample contains peanut traces, the antigen in the antibody-antigen complex is substituted by the peanut protein. (Click to enlarge).</p> | ||
| + | </div> | ||
| + | |||
</div> | </div> | ||
<div class="text-panel two-columns" style="margin-top:0px;padding-top:0px"> | <div class="text-panel two-columns" style="margin-top:0px;padding-top:0px"> | ||
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| - | <div class="image-box" style="width:500px"> | + | <div class="image-box" style="width:500px;display:inline-block;"> |
| - | <a href="# | + | <a href="#fig2" name="modal"> |
<img src="https://static.igem.org/mediawiki/2014/0/09/Linkoping_sweden_GFP_RFP_FRET.png" width="500px" height="auto" title="FRET"></a> | <img src="https://static.igem.org/mediawiki/2014/0/09/Linkoping_sweden_GFP_RFP_FRET.png" width="500px" height="auto" title="FRET"></a> | ||
<div id="boxes"> | <div id="boxes"> | ||
| - | <div id=" | + | <div id="fig2" class="window" style="width:760px;height:310px"> |
<img src="https://static.igem.org/mediawiki/2014/0/09/Linkoping_sweden_GFP_RFP_FRET.png" width="760px" height="310px" title="FRET"> | <img src="https://static.igem.org/mediawiki/2014/0/09/Linkoping_sweden_GFP_RFP_FRET.png" width="760px" height="310px" title="FRET"> | ||
| - | <p> | + | <p>Figure 2. FRET, fluorescence resonance energy transfer is possible if there is a spectral overlap between the emission band of the donor and the excitation band of the acceptor molecule. The fluorescence emission from the donor is decreased followed by a reabsorption of the acceptor. The energy transferred results in an increase in the acceptor emission letting the light be emitted at a longer wavelength.</p> |
<p>FRET can occur if the donor and acceptor are close to each other. This makes it possible to measure the distance between two chromophores. The orientation of the transition dipoles of the donor and acceptor is another criteria.</p> | <p>FRET can occur if the donor and acceptor are close to each other. This makes it possible to measure the distance between two chromophores. The orientation of the transition dipoles of the donor and acceptor is another criteria.</p> | ||
</div> | </div> | ||
<div id="mask"></div> | <div id="mask"></div> | ||
</div> | </div> | ||
| - | <p> | + | <p>Figure 2. FRET, fluorescence resonance energy transfer is possible if there is a spectral overlap between the emission band of the donor and the excitation band of the acceptor molecule. The fluorescence emission from the donor is decreased followed by a reabsorption of the acceptor. The energy transferred results in an increase in the acceptor emission letting the light be emitted at a longer wavelength.</p> |
<p>FRET can occur if the donor and acceptor are close to each other. This makes it possible to measure the distance between two chromophores. The orientation of the transition dipoles of the donor and acceptor is another criteria. (Click to enlarge).</p> | <p>FRET can occur if the donor and acceptor are close to each other. This makes it possible to measure the distance between two chromophores. The orientation of the transition dipoles of the donor and acceptor is another criteria. (Click to enlarge).</p> | ||
| + | </div> | ||
| + | |||
| + | <div class="image-box" style="width:500px;display:inline-block;"> | ||
| + | <a href="#fig3" name="modal"> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/1/16/Linkoping_sweden_Fitc_ensam.png" width="500px" height="auto" title="FRET"></a> | ||
| + | <div id="boxes"> | ||
| + | <div id="fig3" class="window" style="width:760px;height:310px"> | ||
| + | <img src="https://static.igem.org/mediawiki/2014/1/16/Linkoping_sweden_Fitc_ensam.png" width="760px" height="310px" title="FRET"> | ||
| + | <p>Figure 3. If ara h 1 protein is avaliable and the antibody binds to it, FRET will no longer occur. Only FITC will emit photons due to increased distance from RFP.</p> | ||
| + | </div> | ||
| + | <div id="mask"></div> | ||
| + | </div> | ||
| + | <p>Figure 3. If ara h 1 protein is avaliable and the antibody binds to it, FRET will no longer occur. Only FITC will emit photons due to increased distance from RFP. </p> | ||
</div> | </div> | ||
Latest revision as of 18:14, 17 October 2014
The Biology
Our approach is to use an antibody in combination with a fluorescent probe to detect the Ara h1 antigen. This system will have three potential parts: an antibody complex consisting of an polyclonal or monoclonal antibody connected to a fluorescent probe, a default-epitope complex consisting of one or five epitope(s) linked to a fluorescent probe (number of epitopes dependent on antibody class), and the Ara h1 protein. These are potential parts in the sense that the system will always contain the first two parts (antibody complex and the default epitope complex), but will only contain the third part, the Ara h1 protein, if the inserted sample has been contaminated with peanut protein. The biosensor system is to be implemented in a device consisting of a box containing the sensors necessary to detect fluorescence and any eventual changes in fluorescence. This information is then processed by a microprocessor to give a color indication, red or green, correlating to the changes in fluorescence. So how exactly will the system work? Well, there are two possible scenarios for our biosensor system, either the sample does not contain peanut protein (Ara h) (Fig. 1A) or it does (Fig. 1B).
Scenario: No peanut protein contamination
The first scenario is default for our biosensor; it contains two interacting parts in the form of an antibody connected to a fluorescent probe (Fluorescein isothiocyanate, or FITC) and epitope(s) bound to a fluorescent probe (Red Fluorescence Protein or RFP). The epitope(s) bind to the antibody, and when a light source is shone on the biosensor, the incoming light is absorbed by FITC and transferred to RFP through Förster Resonance Energy Transfer (FRET) (Fig. 2). The emitted wavelength is detected and then processed by a microprocessor which makes a LED light-panel shine green. Green light thus indicates that the system only contains the antibody and epitope complexe, and has not been contaminated by peanut protein.
Fig 1A. No peanut protein contamination
Figure 1A. No peanut protein contamination. When the cuvette has been loaded with a sample that doesn't contain any peanut, the antibody-antigen complex remains unchanged. (Click to enlarge).
Scenario: Peanut protein contamination
The other possible scenario is that the inserted food sample has been contaminated by peanut and that the system thus contains Ara h1. The peanut protein will bind with a higher affinity to the FITC-antibody complex, thus forcing the epitope-RFP complex out of the picture. Ara h1 bound to the antibody-FITC complex reduces FRET as applied light is absorbed and emitted solely by FITC. This results in a wavelength change which is detected and processed, causing a LED light-panel switch from green to red. Red light thus indicates that the system has been contaminated by peanut and that the food is unsafe to consume.
Fig 1B. Peanut protein contamination
Figure 1B. Peanut protein contamination. When the loaded food-sample contains peanut traces, the antigen in the antibody-antigen complex is substituted by the peanut protein. (Click to enlarge).
Figure 2. FRET, fluorescence resonance energy transfer is possible if there is a spectral overlap between the emission band of the donor and the excitation band of the acceptor molecule. The fluorescence emission from the donor is decreased followed by a reabsorption of the acceptor. The energy transferred results in an increase in the acceptor emission letting the light be emitted at a longer wavelength.
FRET can occur if the donor and acceptor are close to each other. This makes it possible to measure the distance between two chromophores. The orientation of the transition dipoles of the donor and acceptor is another criteria.
Figure 2. FRET, fluorescence resonance energy transfer is possible if there is a spectral overlap between the emission band of the donor and the excitation band of the acceptor molecule. The fluorescence emission from the donor is decreased followed by a reabsorption of the acceptor. The energy transferred results in an increase in the acceptor emission letting the light be emitted at a longer wavelength.
FRET can occur if the donor and acceptor are close to each other. This makes it possible to measure the distance between two chromophores. The orientation of the transition dipoles of the donor and acceptor is another criteria. (Click to enlarge).
Figure 3. If ara h 1 protein is avaliable and the antibody binds to it, FRET will no longer occur. Only FITC will emit photons due to increased distance from RFP.
Figure 3. If ara h 1 protein is avaliable and the antibody binds to it, FRET will no longer occur. Only FITC will emit photons due to increased distance from RFP.
