Team:ESBS-Strasbourg/proteolux

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<p><br/><a href="http://2010.igem.org/Team:ESBS-Strasbourg">&nbsp;&nbsp;HOME</a></p>
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<p><br/><a href="http://2010.igem.org/Team:ESBS-Strasbourg/proteolux">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HOME&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p>
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<p><br/><a href="http://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p>
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&nbsp;&nbsp;TEAM</a></p>
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Overview</a></li>
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ProteOlux Basic</a></li>
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ProteOlux Plus</a></li>
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Strasbourg</a></li>
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Collaboration</a></li>
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&nbsp;&nbsp;PROJECT</a></p>
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Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p>
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ProteOlux Basic</a></li>
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Visual Description</a></li>
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Acknowledgment</a></li>
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&nbsp;&nbsp;RESULTS</a></p>               
 
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Biobricks</a></li>
 
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Synthetic Photoreceptors</a></li>
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Cancer</a></li>
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HUMAN PRACTICE</a></p>
 
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Organisation</a></li>
 
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Survey</a></li>
 
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The ClpX game</a></li>
 
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Project Safety</a></li>
 
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;SPONSORS</a></p>
 
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OUR SPONSORS
 
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<a href="http://www.oseo.fr/" target="_blank">
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<img src="http://2010.igem.org/wiki/images/d/d1/ESBS-Strasbourg-Oseo.jpg" width="160" height="76" border="0"></a>
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<div style="position: relative; width: 800px; height: 40px; id="layer1" align="justify">
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You are interested in analyzing a specific protein in its natural cell environment? You want to analyze its dynamics, by installing any defined protein concentration in your cell without interfering with the cell’s metabolism? <b>Proteolux</b> offers the solution:</div></center>
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&nbsp;&nbsp;&nbsp;
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<span style="color:ivory;">Words written in <b><font color="#E9AF03">Yellow</font></b>  
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are hyperlinks to other pages.</span>
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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>
<br>
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Let me guide you</span>
 
 +
<center><b>A specific light-controllable protein degradation system for any defined protein.</b></center>
 +
<br><br>
-
+
<center>
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<td width="750" height="400" bgcolor="#414141"  valign="top">
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<div style="position: relative; width: 800px; height: 80px; id="layer1" align="justify">
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<br>
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Proteolux’s aim is to offer new and more sophisticated methods for protein analysis. Proteins are the actual active factors within cells and controlling protein concentrations leads to the control over the cell itself. Protein levels are installed by a complex homeostasis of de-novo protein synthesis and protein degradation. There are already some techniques for controlling specific protein expression. However, up to now, there are only a few systems that offer specific control about protein degradation. Most old techniques use protein knock-out or RNA interference to analyze the protein absence phenotype (figure 1).</div></center>
 +
<br><br>
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<center>
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<div class="heading">
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Project
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</div>
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<br>
<br>
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<div class="desc">
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<img src="http://2010.igem.org/wiki/images/d/d5/ESBS-Strasbourg-Prot-interference.png" width="500px" height="196px"></a>
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The aim of our <b><i><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Project"><font color="#E9AF03">
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</center>
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project</font></a></i></b> 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.
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<div style="position: relative; width: 500px; height: 60px; id="layer1" align="justify">
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<i><font color="#cccccc" size="1">Figure 1: Overview about the synthesis pathway of proteins and the different layers of regulation. The coding information of the DNA is transcribed into mRNA, which is further translated into proteins. The repression of expression protein can take place on three different layers of regulation. Classic methods include gene knockout or RNA interference, which contain several drawbacks. Proteolux defines a new layer of regulation directly on protein level, which can further regulate DNA and mRNA levels.</font></i></a></div>
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<body>
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<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/Vb1Y7lsKEho?hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/Vb1Y7lsKEho?hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object>
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<br><br>
<br><br>
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<center>
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<div style="position: relative; width: 800px; height: 350px; id="layer1" align="justify">
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Both methods exhibit several disadvantages. Gene knockout allows only the analysis of complete protein absence phenotypes, which creates problems in the case of essential proteins, whose absence is lethal to cells. Additionally, the creation of knock out strains (e.g. knock out mouse) for in-vivo analysis demands 3 generations and therefore in advance preparation of the experiment. RNA interference allows more specific regulation of protein expression than gene knockout. However, the main problems are the delivery of the RNAi, a design that is specific in order to avoid off-target effects. Moreover, the actual amount of protein in the cell cannot be controlled as the silencing percentage differs from interfering RNA to interfering RNA and it is difficult to achieve total silencing.  Other disadvantages are the limited duration of the repression and toxic effects on cells.
-
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<br>
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ESBS-Strasbourg Team
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</div>
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<br><br>
<br><br>
-
 
+
Proteolux’s specific light-controllable protein degradation system interacts directly on protein level, which allows complete control over the protein level as well as the over DNA and RNA layers. The Proteolux system offers the solution for the drawbacks of the other methods. The extremely high controllability allows fine regulation of any protein concentration, which allows also analysis of essential proteins. Different protein concentrations can be installed over time on demand of the experimenter. In-vivo experiments can be made directly after transfection with the system. Long preparation time before conducting experiments is not needed. The protein can be analyzed in its native conformation, as there is no tagging or fusion with other markers necessary. Furthermore, the metabolism of the cell is not altered, as the system is completely controlled by light. There is no off-target degradation due to the extreme high specificity of the interaction partners of the system. Moreover, the Proteolux system does not exhibit any toxicity to the cells.  
-
<div class="desc">
+
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<img border="3"  src="http://2008.igem.org/wiki/images/c/c5/Esbs.jpg" width="120" height="240" align="left" hspace="50">
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<br><br>
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We are a student-run team from the trinational graduate school <i><font color="#8888FF">
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&quot;École supérieur de Biotechnologie Strasbourg&quot;</font></i> <a href="http://www-esbs.u-strasbg.fr"><font color="#E9AF03">
+
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(ESBS)</font></a> located in the capital of Europe - <b>Strasbourg, France.</b>
+
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The ESBS is a joint-venture of the Universities of Freiburg and Karlsruhe in
+
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Germany, the University of Bale in Switzerland and the University of Strasbourg
+
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in France. Students come from all four partner universities and furthermore from
+
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all around the three countries. </p>
+
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</div>
+
<br><br>
<br><br>
 +
The light control of the Proteolux systems provides multiple advantages over the use of classical chemical inducers. The system responds instantly to the light signal, which can be used to specifically regulate its action. Chemical inducers do not respond immediately, due to the diffusion time to get to the place of action. Once the chemical inducer at its side of action it is not possible to turn the system off again. However, light offers this switchable control (on and off) of the system. Furthermore, light does not interact with the host metabolism which allows protein analysis in its unchanged natural environment. Another great advantage of light over chemical molecules is its inexpensiveness and its unlimited availability (summarized in the table below).
 +
</div></center>
 +
<br>
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<div class="desc">
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<center>
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This is the second year that the <b>ESBS-Strasbourg</b> participates in the iGEM
+
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competition. Our team consists of <i>12 students</i> coming from different
+
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scientific backgrounds prior to the ESBS. This will help us to deal with the
+
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difficulties coming up during this interdisciplinary project. Our shared
+
-
education at the ESBS however enables us to communicate more easily with each
+
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other. An overview of the people involved and our competences can be found on
+
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the <b><i><a href="http://2010.igem.org/Team:ESBS-Strasbourg/Team"><font color="#E9AF03">
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team</font></a></i></b> page.
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<div class="heading">
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<img src="http://2010.igem.org/wiki/images/e/ef/ESBS-Strasbourg-advantage.png" width="500px" height="209px"></a>
-
iGEM - INTERNATIONAL GENETICALLY ENGINEERED MACHINES
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</center>
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<br><br>
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<div class="desc">
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<b>iGEM</b> (international Genetically Engineered Machines) is a competition
+
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organized by MIT (Massachusetts Institute of Technology) in <b>Boston, USA</b>,
+
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since 2004 and has become one of the largest international competitions in the
+
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field of science. This year 128 teams of undergraduate students compete against
+
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each other, and for the second time <b>ESBS-Strasbourg</b> team is joining this
+
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competition.  
+
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<br><br>
+
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<b>Synthetic biology</b> is a new area of biological research that combines <i>
+
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science</i> and <i>engineering</i>. Synthetic biology encompasses a variety of
+
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different approaches, methodologies and disciplines. We can define the synthetic
+
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biology as the <u>design</u> and<u> (re)construction</u> of new <u>biological
+
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functions</u> and <u>systems</u> not found in nature.
+
<br><br>
<br><br>
<center>
<center>
-
<img src="http://2010.igem.org/wiki/images/f/fa/Car-parts-.png">
+
<b>We sparked your interest in Proteolux? Please follow the guide to learn more about the scientific background of our system.</b></center>
 +
<br>
 +
<center>
 +
<a target="_blank" href="http://2010.igem.org/Team:ESBS-Strasbourg/proteolux/guide">
 +
<img src="http://2010.igem.org/wiki/images/1/10/ESBS-Strasbourg-guide.gif" height="100px" width="82.4px"></a>
</center>
</center>
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<br><br>
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<center><b>Let me guide you !</b></center>
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Similar to the construction of a <b><i><font color="#8888FF">car</font></i></b>,
+
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Synthetic Biology uses simple <b><i> <font color="#8888FF">gene building blocks</font></i></b>
+
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for the construction of new complex <b> systems</b> with distinct functions.
+
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These gene building blocks are collected in a database by <b> iGEM</b> and can
+
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be used by all participants of the competition. So far, the collection contains
+
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more than <i><a href="http://partsregistry.org/cgi/partsdb/Statistics.cgi "><font color="#E9AF03">
+
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13,000 gene building blocks</font></a></i>, due to the continuous development of
+
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new parts over the last years. This summer all teams work on self-developed
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projects which will then be presented in the beginning of November at the
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“Jamboree” in Boston. Several prizes in different categories will be awarded.
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Latest revision as of 21:59, 29 November 2010

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ESBS-Strasbourg


ProteOlux


You are interested in analyzing a specific protein in its natural cell environment? You want to analyze its dynamics, by installing any defined protein concentration in your cell without interfering with the cell’s metabolism? Proteolux offers the solution:

A specific light-controllable protein degradation system for any defined protein.


Proteolux’s aim is to offer new and more sophisticated methods for protein analysis. Proteins are the actual active factors within cells and controlling protein concentrations leads to the control over the cell itself. Protein levels are installed by a complex homeostasis of de-novo protein synthesis and protein degradation. There are already some techniques for controlling specific protein expression. However, up to now, there are only a few systems that offer specific control about protein degradation. Most old techniques use protein knock-out or RNA interference to analyze the protein absence phenotype (figure 1).



Figure 1: Overview about the synthesis pathway of proteins and the different layers of regulation. The coding information of the DNA is transcribed into mRNA, which is further translated into proteins. The repression of expression protein can take place on three different layers of regulation. Classic methods include gene knockout or RNA interference, which contain several drawbacks. Proteolux defines a new layer of regulation directly on protein level, which can further regulate DNA and mRNA levels.


Both methods exhibit several disadvantages. Gene knockout allows only the analysis of complete protein absence phenotypes, which creates problems in the case of essential proteins, whose absence is lethal to cells. Additionally, the creation of knock out strains (e.g. knock out mouse) for in-vivo analysis demands 3 generations and therefore in advance preparation of the experiment. RNA interference allows more specific regulation of protein expression than gene knockout. However, the main problems are the delivery of the RNAi, a design that is specific in order to avoid off-target effects. Moreover, the actual amount of protein in the cell cannot be controlled as the silencing percentage differs from interfering RNA to interfering RNA and it is difficult to achieve total silencing. Other disadvantages are the limited duration of the repression and toxic effects on cells.

Proteolux’s specific light-controllable protein degradation system interacts directly on protein level, which allows complete control over the protein level as well as the over DNA and RNA layers. The Proteolux system offers the solution for the drawbacks of the other methods. The extremely high controllability allows fine regulation of any protein concentration, which allows also analysis of essential proteins. Different protein concentrations can be installed over time on demand of the experimenter. In-vivo experiments can be made directly after transfection with the system. Long preparation time before conducting experiments is not needed. The protein can be analyzed in its native conformation, as there is no tagging or fusion with other markers necessary. Furthermore, the metabolism of the cell is not altered, as the system is completely controlled by light. There is no off-target degradation due to the extreme high specificity of the interaction partners of the system. Moreover, the Proteolux system does not exhibit any toxicity to the cells.

The light control of the Proteolux systems provides multiple advantages over the use of classical chemical inducers. The system responds instantly to the light signal, which can be used to specifically regulate its action. Chemical inducers do not respond immediately, due to the diffusion time to get to the place of action. Once the chemical inducer at its side of action it is not possible to turn the system off again. However, light offers this switchable control (on and off) of the system. Furthermore, light does not interact with the host metabolism which allows protein analysis in its unchanged natural environment. Another great advantage of light over chemical molecules is its inexpensiveness and its unlimited availability (summarized in the table below).




We sparked your interest in Proteolux? Please follow the guide to learn more about the scientific background of our system.

Let me guide you !