Team:ESBS-Strasbourg/Project

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ESBS - Strasbourg
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</li>
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<li>
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<p><br/><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team">&nbsp;&nbsp;TEAM</a></p>
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<p><br/><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team">
 +
&nbsp;&nbsp;TEAM</a></p>
<ul>
<ul>
-
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team">Overview</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team">
 +
Overview</a></li>
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team#under">
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team#under">
Students</a></li>
Students</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team#instructors">
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team#advisors">
Advisors</a></li>
Advisors</a></li>
 +
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team#instructors">
 +
Instructors</a></li>
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team#uni">
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team#uni">
Strasbourg</a></li>
Strasbourg</a></li>
 +
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team#collaboration">
 +
Collaboration</a></li>
                                
                                
</ul>
</ul>
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<li>
<li>
<p><br />
<p><br />
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project">&nbsp;&nbsp;PROJECT</a></p>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project">
 +
&nbsp;&nbsp;PROJECT</a></p>
<ul>
<ul>
-
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project">Overview</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project">
 +
Overview</a></li>
                                 <li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Strategy">
                                 <li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Strategy">
Strategy</a></li>
Strategy</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Acknowledgment">Acknowledgment</a></li>
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                                <li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/visual">
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Visual Description</a></li>
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                                <li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Application">
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Application</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Acknowledgment">
 +
Acknowledgment</a></li>
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">
Reference</a></li>
Reference</a></li>
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</li>
</li>
<li>
<li>
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<p><br/><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Results">&nbsp;&nbsp;RESULTS</a></p>                 
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<p><br/><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Results/Biobricks">
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&nbsp;&nbsp;RESULTS</a></p>                 
                         <ul>
                         <ul>
-
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Results/Biobricks">Biobricks</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Results/Biobricks">
 +
Biobricks</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Results/Assembly">
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<font size="3">Biobrick Assembly Technique</font></a></li>
                                 <li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Results/Characterization">
                                 <li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Results/Characterization">
Characterization</a></li>
Characterization</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Results/Modelling">Modelling</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Results/Modelling">
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Modeling</a></li>
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</li>
<li>
<li>
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<p><br/><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Notebook">&nbsp;&nbsp;NOTEBOOK</a></p>
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<p><br/><a>
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&nbsp;&nbsp;NOTEBOOK</a></p>
                         <ul>
                         <ul>
-
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Notebook/Syntethic">Synthetic Photoreceptors</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Notebook/Syntethic">
 +
Synthetic Photoreceptors</a></li>
                                 <li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Notebook/Microfluidics">
                                 <li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Notebook/Microfluidics">
Microfluidics</a></li>
Microfluidics</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Notebook/Labbook">Lab-book</a></li>
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                              <li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Results/Device">Lighting device</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Notebook/Labbook">
 +
Lab-book</a></li>
</ul>
</ul>
</li>
</li>
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 +
<li>
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<p><br/><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice">
 +
HUMAN PRACTICE</a></p>
 +
                        <ul>
 +
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#organisation">
 +
Organisation</a></li>
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<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#survey">
 +
Survey</a></li>
 +
                                <li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#video">
 +
The ClpX video</a></li>
 +
                                <li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#game">
 +
The ClpX game</a></li>
 +
<li><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#safety">
 +
Project Safety</a></li>
 +
 +
</ul>
 +
 +
</li>
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                 <li class="last">
                 <li class="last">
<p><br />
<p><br />
-
<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Sponsors">&nbsp;&nbsp;SPONSORS</a></p>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Sponsors">
 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;SPONSORS</a></p>
</li>
</li>
</ul>
</ul>
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</div>
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<div class="desc">
<div class="desc">
Our device construction is divided into 4 components :<br><br>
Our device construction is divided into 4 components :<br><br>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Protease">1. Degradation system</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Strategy#degradation">1. Degradation system</a>
<br>
<br>
-
<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Light">2. Light detection system</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Strategy#light">2. Light detection system</a>
<br>
<br>
-
<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Tagging">3. Protein Tagging</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Strategy#tagging">3. Protein Tagging</a>
<br>
<br>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/System">4. Light controllable protease</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project/Strategy#system">4. Light controllable protease</a>
<br><br>
<br><br>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/notebook">Notebook</a>
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/Results/Biobricks">Biobricks</a>
</div>
</div>
</p></div>
</p></div>
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<span style="color:ivory;">
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&nbsp;&nbsp;
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<a href="http://2010.igem.org/Team:ESBS-Strasbourg/science">
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<img border="0" src="http://2010.igem.org/wiki/images/d/da/ESBS-Strasbourg-Clpx.gif" width="70" height="85" ></a>
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<br>
 +
Let me guide you</span>
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<div class="heading">A light-controllable specific protein degradation system as new standard for synthetic biology</div>
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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 new component that could be universally used to build more complex or more controllable biological circuits inside chassis organisms. This new component consists in a protease that can be reversibly induced by light, after which it can specifically degrade any given target protein as long as it is properly tagged.
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<br>
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This system is to be constitutively expressed in the chassis, but will remain as an inactive state until light application. The protease activity is thus expected to be instantly turned on upon application of light since there is neither transcriptional nor translationnal delay.
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<br><br>
<br><br>
-
<p><b>Background:</b></p>
 
<br>
<br>
-
The system combines two important different features : specificity, and light sensitivity.
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-
<br>
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<p><b>Theoretical Background:</b></p>
-
<br>
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-
-Specificity : We based our system on the ClpXP protease, from E. coli. In wild type E. coli, ClpX forms a ring of hexamer and binds to a double heptamer of ClpP. ClpX recognizes a specific C-terminal degradation tag called SsrA and starts to unfold the tagged protein. The denatured polypeptide is translocated into the ClpP subunit, which breaks peptide bonds.
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<br>
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<br>
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-
Note : In vivo, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell.
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<br>
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<br>
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-
-Light sensitivity : We used PhyB (phytochrome B) from A. thaliana and its natural chromophore PCB (phycocyanobilline). Holo phytochrome can absorb red light (pick at 660 nm) and through a structural transition it reaches its activated Pfr state. Under far red light (730 nm) the holo phytochrome goes back to its fundamental inactive Pr state. PIF-3 and PIF-6 (Phytochrome Interacting Factor 3 and 6) are natural partners of PhyB in A. thaliana and bind to PhyB in the Pfr state but not in the Pr state. This is the basis of the controllable feature of our system.
+
<br>
<br>
 +
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>
 +
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>
 +
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>
 +
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>
-
<p><b>How does it work?</b></p>
+
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>In our system this adaptator will be replaced by a light sensitive tethering system.</b></p>
-
There are different variant of SsrA tags which are more or less efficiently recognized by ClpX. The DAS tag (T.Baker et al.) has a higher Kd value than wild type SsrA tag, and thus degradation of DAS-tagged proteins is not significant
+
-
<br>
+
-
<br>
+
-
In vivo though, DAS-tagged protein can still be significantly degraded within the range of physiological concentrations under the action of an adaptator protein (SspB) which help tethering the tagged protein to the protease.
+
-
<br>
+
-
<br>
+
-
The idea is to replace this adaptator system with a light sensitive tethering system.
+
<br>
<br>
<br><br>
<br><br>
-
<p><b>How did we proceed?</b></p>
 
-
<br>
 
-
 
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For the protease, we fused either the 650 or 900 first aminoacids of PhyB to a trimer of the last 364 aminoacids of ClpX. Indeed, the N terminal end of ClpX contains a domain which is responsible for interaction with its natural adaptator protein (SspB)  that we would avoid. As this N-terminal domain is also partially responsible for ClpX subunits complexation into an hexamer, fusing three C-terminal end of ClpX together with appropriate linkers increases the stability of the system (T. Baker et al.) in the absence of this N-terminal domain.
 
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<br>
 
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<br>
 
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The target protein will be tagged with both DAS at C-terminal end, and with  the Active Phytochrome Binding (APB) motif (the first hundred aminoacids of PIF-3 or PIF-6), either at N terminal end or just upstream the DAS tag.
 
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<br>
 
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<br>
 
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Under red light, the APB motif of the target protein binds to PhyB and facilitate recognition of DAS tag by ClpX, thus promoting degradation.
 
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Far red light instantly inhibits degradation of tagged proteins.
 
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<br>
 
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</div>
</div>
</td>
</td>

Latest revision as of 22:57, 27 October 2010

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ESBS - Strasbourg



PROJECT INDEX

  
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A light-controllable specific protein degradation system as new standard for synthetic biology





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.