Team:ESBS-Strasbourg/Project
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Modelling"> | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Modelling"> | ||
Modeling</a></li> | Modeling</a></li> | ||
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</ul> | </ul> | ||
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Notebook/Microfluidics"> | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Notebook/Microfluidics"> | ||
Microfluidics</a></li> | Microfluidics</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Device">Lighting device</a></li> | ||
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Notebook/Labbook"> | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Notebook/Labbook"> | ||
Lab-book</a></li> | Lab-book</a></li> | ||
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<li> | <li> | ||
- | <p><br/><a> | + | <p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice"> |
HUMAN PRACTICE</a></p> | HUMAN PRACTICE</a></p> | ||
<ul> | <ul> | ||
- | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice | + | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#organisation"> |
Organisation</a></li> | Organisation</a></li> | ||
- | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice | + | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#survey"> |
Survey</a></li> | Survey</a></li> | ||
- | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice | + | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#video"> |
The ClpX video</a></li> | The ClpX video</a></li> | ||
- | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice | + | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#game"> |
The ClpX game</a></li> | The ClpX game</a></li> | ||
- | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/ | + | <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#safety"> |
Project Safety</a></li> | Project Safety</a></li> | ||
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<a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Strategy#system">4. Light controllable protease</a> | <a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Strategy#system">4. Light controllable protease</a> | ||
<br><br> | <br><br> | ||
- | <a href="https://2010.igem.org/Team:ESBS-Strasbourg/ | + | <a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Biobricks">Biobricks</a> |
</div> | </div> | ||
</p></div> | </p></div> | ||
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<td width="10" rowspan=3 bgcolor="#222222"> | <td width="10" rowspan=3 bgcolor="#222222"> | ||
</div> | </div> | ||
+ | <div id="windowbox" style="position:fixed; top:50%; left:20px; width:11%;"> | ||
+ | <span style="color:ivory;"> | ||
+ | | ||
+ | <a href="https://2010.igem.org/Team:ESBS-Strasbourg/science"> | ||
+ | <img border="0" src="https://static.igem.org/mediawiki/2010/d/da/ESBS-Strasbourg-Clpx.gif" width="70" height="85" ></a> | ||
+ | <br> | ||
+ | Let me guide you</span> | ||
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<br> | <br> | ||
<body> | <body> | ||
- | <object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/ | + | |
+ | <object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/9L2nenFWdM0?fs=1&hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/9L2nenFWdM0?fs=1&hl=en_US" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object> | ||
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</body> | </body> | ||
</center> | </center> | ||
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<div class="desc"> | <div class="desc"> | ||
<br> | <br> | ||
- | + | The aim of our project is to engineer a light-inducible degradation system as a new fundamental component that can be easily used to build more complex biological circuits inside chassis organisms. This new component consists of the bacterial protease ClpXP from Escherichia Coli fused to the photoreceptor protein phytochrome B of Arabidopsis thaliana. The degradation system is universally applicable to any given protein by addition of a specially designed Biobrick containing the phytochrome interacting factor (PIF3/6) and a specific degradation sequence (DAS-tag). This Biobrick can be added to the C-terminal of the target protein by standard assembly methods. Illumination of red light (660nm) induces a conformational change in phytochrome B and activates the system, an impulse of far-red light (730nm) leads to disruption of the degradation. This allows a tight control over the catalytic activity, enabling the modulation of protein function in a general fashion with the combined characteristics of specificity, high temporal precision and rapid reversibility. | |
- | The aim of our project is to engineer a light-inducible degradation system as a new fundamental component that can be easily used to build more complex biological circuits inside chassis organisms. This new component consists of the bacterial protease ClpXP from Escherichia Coli fused to the photoreceptor protein phytochrome B of Arabidopsis thaliana. The degradation system is universally applicable to any given protein by | + | |
<br><br> | <br><br> | ||
<br> | <br> | ||
- | + | ||
<p><b>Theoretical Background:</b></p> | <p><b>Theoretical Background:</b></p> | ||
<br> | <br> | ||
- | Our system contains several parts: the bacterial ClpXP protease from E. Coli | + | Our system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor (PIF3/6) from A. thaliana for the light-dependent part of the system. |
<br><br> | <br><br> | ||
- | + | The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. | |
- | + | ||
- | + | ||
- | + | ||
<br><br> | <br><br> | ||
- | + | PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm)PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused tothe N terminus of a trimeric form of ClpX-N in which the subunits were connected with a flexible linker to stabilize the enzyme (thanks to the findings of Baker & Sauer in 2005). | |
<br><br> | <br><br> | ||
- | + | Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal. | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<br><br> | <br><br> | ||
- | To engineer controlled degradation, Baker and Sauer (2006) designed a series of modified | + | In the native organism, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell. To engineer controlled degradation, Baker and Sauer (2006) designed a series of modified SsrA tags that have weakened interactions with ClpXP. The DAS-tag presents one of these artificial sequence; its Kd value is significantly higher than the one of wild type SsrA, thus degradation of DAS-tagged proteins is not significant within the range of physiological concentrations. However, through the action of the adaptor protein SspB which helps tethering the tagged protein to the protease, DAS-tagged proteins are significantly degraded. |
<br><br> | <br><br> | ||
- | <p><b> | + | <p><b>In our system this adaptator will be replaced by a light sensitive tethering system.</b></p> |
<br> | <br> | ||
<br><br> | <br><br> |
Latest revision as of 22:57, 27 October 2010
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Abstract
The aim of our project is to engineer a light-inducible degradation system as a new fundamental component that can be easily used to build more complex biological circuits inside chassis organisms. This new component consists of the bacterial protease ClpXP from Escherichia Coli fused to the photoreceptor protein phytochrome B of Arabidopsis thaliana. The degradation system is universally applicable to any given protein by addition of a specially designed Biobrick containing the phytochrome interacting factor (PIF3/6) and a specific degradation sequence (DAS-tag). This Biobrick can be added to the C-terminal of the target protein by standard assembly methods. Illumination of red light (660nm) induces a conformational change in phytochrome B and activates the system, an impulse of far-red light (730nm) leads to disruption of the degradation. This allows a tight control over the catalytic activity, enabling the modulation of protein function in a general fashion with the combined characteristics of specificity, high temporal precision and rapid reversibility. Theoretical Background: Our system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor (PIF3/6) from A. thaliana for the light-dependent part of the system. The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm)PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused tothe N terminus of a trimeric form of ClpX-N in which the subunits were connected with a flexible linker to stabilize the enzyme (thanks to the findings of Baker & Sauer in 2005). Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal. In the native organism, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell. To engineer controlled degradation, Baker and Sauer (2006) designed a series of modified SsrA tags that have weakened interactions with ClpXP. The DAS-tag presents one of these artificial sequence; its Kd value is significantly higher than the one of wild type SsrA, thus degradation of DAS-tagged proteins is not significant within the range of physiological concentrations. However, through the action of the adaptor protein SspB which helps tethering the tagged protein to the protease, DAS-tagged proteins are significantly degraded. In our system this adaptator will be replaced by a light sensitive tethering system. |