Team:ESBS-Strasbourg/Results/Biobricks

From 2010.igem.org

(Difference between revisions)
 
(33 intermediate revisions not shown)
Line 107: Line 107:
position: absolute;
position: absolute;
top: -20px;
top: -20px;
-
font: 18px Trebuchet MS;
+
font: 17px Trebuchet MS;
text-align: center;
text-align: center;
           }
           }
Line 138: Line 138:
</>
</>
</div>
</div>
 +
<div id="content0" style="display:none">
<div id="content0" style="display:none">
Line 201: Line 202:
-
</div>
 
-
 
<div class="menubg">
<div class="menubg">
</div>
</div>
Line 257: Line 256:
position: relative;
position: relative;
float: left;
float: left;
-
width:170px;
+
width:143px;
height:75px;
height:75px;
color:#fff;
color:#fff;
Line 274: Line 273:
border:none;
border:none;
height:193px;
height:193px;
-
width:193px;
+
width:153px;
background:#000;
background:#000;
  /*opacity for IE5+*/
  /*opacity for IE5+*/
Line 303: Line 302:
ul#nav li.last,ul#nav li.last ul{border:none;width:193px;}
ul#nav li.last,ul#nav li.last ul{border:none;width:193px;}
   
   
-
</style>  
+
</style>
 +
<div class="outer">
<div class="outer">
Line 313: Line 313:
</li>
</li>
<li>
<li>
-
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Team">&nbsp;&nbsp;TEAM</a></p>
+
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Team">
 +
&nbsp;&nbsp;TEAM</a></p>
<ul>
<ul>
-
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Team">Overview</a></li>
+
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Team">
 +
Overview</a></li>
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Team#under">
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Team#under">
Students</a></li>
Students</a></li>
-
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Team#instructors">
+
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Team#advisors">
Advisors</a></li>
Advisors</a></li>
 +
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Team#instructors">
 +
Instructors</a></li>
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Team#uni">
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Team#uni">
Strasbourg</a></li>
Strasbourg</a></li>
 +
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Team#collaboration">
 +
Collaboration</a></li>
                                
                                
</ul>
</ul>
Line 327: Line 333:
<li>
<li>
<p><br />
<p><br />
-
<a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project">&nbsp;&nbsp;PROJECT</a></p>
+
<a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project">
 +
&nbsp;&nbsp;PROJECT</a></p>
<ul>
<ul>
-
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project">Overview</a></li>
+
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project">
 +
Overview</a></li>
                                 <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Strategy">
                                 <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Strategy">
Strategy</a></li>
Strategy</a></li>
 +
                                <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/visual">
 +
Visual Description</a></li>
                                 <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Application">
                                 <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Application">
Application</a></li>
Application</a></li>
-
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Acknowledgment">Acknowledgment</a></li>
+
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Acknowledgment">
 +
Acknowledgment</a></li>
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">
Reference</a></li>
Reference</a></li>
Line 341: Line 352:
</li>
</li>
<li>
<li>
-
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results">&nbsp;&nbsp;RESULTS</a></p>                 
+
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Biobricks">
 +
&nbsp;&nbsp;RESULTS</a></p>                 
                         <ul>
                         <ul>
-
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Biobricks">Biobricks</a></li>
+
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Biobricks">
 +
Biobricks</a></li>
 +
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Assembly">
 +
<font size="3">Biobrick Assembly Technique</font></a></li>
                                 <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Characterization">
                                 <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Characterization">
Characterization</a></li>
Characterization</a></li>
-
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Modelling">Modelling</a></li>
+
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Modelling">
 +
Modeling</a></li>
 +
 
</ul>
</ul>
Line 353: Line 370:
</li>
</li>
<li>
<li>
-
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Notebook">&nbsp;&nbsp;NOTEBOOK</a></p>
+
<p><br/><a>
 +
&nbsp;&nbsp;NOTEBOOK</a></p>
                         <ul>
                         <ul>
-
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Notebook/Syntethic">Synthetic Photoreceptors</a></li>
+
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Notebook/Syntethic">
 +
Synthetic Photoreceptors</a></li>
                                 <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/Notebook/Labbook">Lab-book</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">
 +
Lab-book</a></li>
</ul>
</ul>
</li>
</li>
 +
 +
<li>
 +
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice">
 +
HUMAN PRACTICE</a></p>
 +
                        <ul>
 +
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#organisation">
 +
Organisation</a></li>
 +
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#survey">
 +
Survey</a></li>
 +
                                <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#video">
 +
The ClpX video</a></li>
 +
                                <li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#game">
 +
The ClpX game</a></li>
 +
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Humanpractice#safety">
 +
Project Safety</a></li>
 +
 +
</ul>
 +
 +
</li>
 +
                 <li class="last">
                 <li class="last">
<p><br />
<p><br />
-
<a href="https://2010.igem.org/Team:ESBS-Strasbourg/Sponsors">&nbsp;&nbsp;SPONSORS</a></p>
+
<a href="https://2010.igem.org/Team:ESBS-Strasbourg/Sponsors">
 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;SPONSORS</a></p>
</li>
</li>
</ul>
</ul>
 +
</div>
 +
</div>
</div>
</div>
</div>
</div>
Line 453: Line 497:
</ul>
</ul>
-
<a href="http://">other link if needed</a>
+
<a href="https://2010.igem.org/Team:ESBS-Strasbourg/Results/Assembly">Our Biobrick Assembly Technique</a>
</div>
</div>
</p></div>
</p></div>
Line 459: Line 503:
<td width="10" rowspan=40 bgcolor="#222222">
<td width="10" rowspan=40 bgcolor="#222222">
</div>
</div>
-
 
+
<div id="windowbox" style="position:fixed; top:50%; left:20px; width:11%;">
 +
<span style="color:ivory;">
 +
&nbsp;&nbsp;
 +
<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>
<td width="750" bgcolor="#414141">
<td width="750" bgcolor="#414141">
<div class="desc">
<div class="desc">
Line 579: Line 629:
<td width="302"><font color="#FFFFFF"><a href="#PHYB642-CLPX3">PhyB642-(linker-&#8710;NClpX)3</font></td>
<td width="302"><font color="#FFFFFF"><a href="#PHYB642-CLPX3">PhyB642-(linker-&#8710;NClpX)3</font></td>
<td width="223"><font color="#FFFFFF">pSB1C3 / Chloramphenicol</font></td>
<td width="223"><font color="#FFFFFF">pSB1C3 / Chloramphenicol</font></td>
-
<td width="187" bgcolor="#009933"><font color="#FFFFFF">Sequenced</font></td>
+
<td width="187" bgcolor="#6600CC"><font color="#FFFFFF">Assembled</font></td>
</tr>
</tr>
<tr>
<tr>
Line 588: Line 638:
</td>
</td>
<td width="223"><font color="#FFFFFF">pSB1C3 / Chloramphenicol</font></td>
<td width="223"><font color="#FFFFFF">pSB1C3 / Chloramphenicol</font></td>
-
<td width="187" bgcolor="#009933"><font color="#FFFFFF">Sequenced</font></td>
+
<td width="187" bgcolor="#6600CC"><font color="#FFFFFF">Assembled</font></td>
</tr>
</tr>
<tr>
<tr>
Line 604: Line 654:
<td width="302"><font color="#FFFFFF"><a href="#NCLPX-L">&#8710;NClpX-linker-&#8710;NClpX-linker-&#8710;NClpX</font></td>
<td width="302"><font color="#FFFFFF"><a href="#NCLPX-L">&#8710;NClpX-linker-&#8710;NClpX-linker-&#8710;NClpX</font></td>
<td width="223"><font color="#FFFFFF">pSB1C3 / Chloramphenicol</font></td>
<td width="223"><font color="#FFFFFF">pSB1C3 / Chloramphenicol</font></td>
-
<td width="187" bgcolor="#009933"><font color="#FFFFFF">Sequenced</font></td>
+
<td width="187" bgcolor="#6600CC"><font color="#FFFFFF">Assembled</font></td>
</tr>
</tr>
<tr>
<tr>
Line 612: Line 662:
<td width="302"><font color="#FFFFFF"><a href="L-NCLPX">(linker-&#8710;NClpX)3 </font></td>
<td width="302"><font color="#FFFFFF"><a href="L-NCLPX">(linker-&#8710;NClpX)3 </font></td>
<td width="223"><font color="#FFFFFF">pSB1C3 / Chloramphenicol</font></td>
<td width="223"><font color="#FFFFFF">pSB1C3 / Chloramphenicol</font></td>
-
<td width="187" bgcolor="#009933"><font color="#FFFFFF">Sequenced</font></td>
+
<td width="187" bgcolor="#6600CC"><font color="#FFFFFF">Assembled</font></td>
</tr>
</tr>
</table>
</table>
Line 637: Line 687:
</td>
</td>
</tr>
</tr>
-
 
<tr>
<tr>
<td width="750" bgcolor="#414141"">
<td width="750" bgcolor="#414141"">
<br>
<br>
<div class="heading">
<div class="heading">
-
<a name="PIF3"></a>
+
<a name="PHYB908"></a>
-
Phytochrome Interacting Factor-3 (PIF3) - <a target="_blank" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K365000"><font color="#E9AF03">BBa_K365000</font></a>
+
Phytochrome B (aa 1-908) - <a target="_blank" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K365002"><font color="#E9AF03">BBa_K365002</font></a>
</div>
</div>
<div class="desc">
<div class="desc">
Line 649: Line 698:
<br>
<br>
<p><b>Background:</b></p>
<p><b>Background:</b></p>
-
PIF3 is a downstream transcription factor in a well studied signalling pathway of A. thaliana, upon stimulation with red (650 nm) light, it binds directly to PhyB and translocates to the nucleus as a heterodimer where it modulates the transcription of response genes. PIF3 binds only the red-light-exposed form of phytochrome, Pfr, and shows no-measurable binding affinity for the dark- or infrared-exposed Pr state.<br>
+
Phytochromes characterised by a red/far-red photochromicity. Through red-light (650–670 nm) absorption the phytochrome undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of the PIF. This light-sensitive interaction has been mapped to the 650-residue amino-terminal photosensory core of PhyB <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[30]</a></i>. The process is completely reversible through absorption in the near infra-red spectrum (705–740 nm).  
-
In our system target proteins are fused to PIF3 and tagged with the DAS degradation sequence which, through light activation, brings the degradation tag in proximity to ClpX.<br>
+
<br><br>
-
<br>
+
The photoreceptor protein PhyB serves for the light-dependent activation of the system, therefore it will be fused to the N-teminal of the ClpX-trimer.
 +
<br><br>
<p><b>Conception:</b></p>
<p><b>Conception:</b></p>
-
The light-sensitive interaction with PhyB has been mapped to the first 100-residue N-terminal activated phytochrome binding (APB) domain of PIF3 <a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">(Lim & Voigt, 2009.)</a><br>
+
In in-vivo applications it has been shown that the PIF-interaction with the PhyB photosensory core (residues 1–650) is irreversible in infrared light. <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[42]</a></i> demonstrated by assaying PIF6 (which has the strongest interactions of all previously reported PIF domains) against different variants of PhyB that the tandem C-terminal PAS domains (residues 1-908)of plant phytochromes are necessary to confer rapid photoreversibility under infrared light.
-
We chose this sequence, as it has already been successfully used in different synthetic in vitro applications that benefitted from its light-sensitive interactions with PhyB. The original sequence contains an XbaI restriction site.
+
The original sequence contains a SpeI restriction within the first 908 residues.
<br><br>
<br><br>
<center>
<center>
-
<img src="https://static.igem.org/mediawiki/2010/1/13/ESBS-Strasbourg-Gene.png" width="100px" height="42px" border="0">
+
<img src="https://static.igem.org/mediawiki/2010/b/b4/ESBS-Strasbourg-PhyB900.png" width="250px" height="167px" border="0">
</center>
</center>
-
<center>
 
-
<i><font size="2">PIF3</font></i>
 
-
</center>
 
-
<br>
 
-
The plasmid containing the PIF3-sequence was provided by <a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Acknowledgment#kircher">the laboratory of Stephan Kircher</a> from the University of Freiburg. For the synthesis of the BioBrick part primers containing the sites of the Fusion Protein BioBrick Assembly Standard were used.
 
<br><br>
<br><br>
-
<b>Forward primer (5’->3’): 51 bp</b>
+
The plasmid containing the PhyB-sequence was provided by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Acknowledgment#weber">the laboratory of Wilfried Weber</a></i> from the University of Freiburg. To create the BioBrick part the sequence was amplified with primers containing the standard prefix with ATG and the fusion suffix of the Fusion Protein Assembly Standard.
 +
<br><br> 
 +
<b>Forward primer (5’->3’): (41bp)</b>
<br>
<br>
-
<span>
+
GGATCC<span style="color:red">gaattc</span><span style="color: #B8CCE4">gcggccgc</span>t<span style="color:#00B050">tctag</span>ATGGTTTCCGGAGTC
-
GGATCC<span style="color:red">gaattc</span><span style="color: #B8CCE4">gcggccgc</span>t<span style="color:#00B050">tctaga</span>tg<b><span style="color:#009999">gccggc</span></b>ATGCCTCTGTTTGAGC</span></p>
+
<br><br>
<br><br>
-
<b>Reverse primer (5’->3’): 51 bp</b>
+
<b>Reverse primer (5’->3’): (52 bp)</b>
<br>
<br>
-
<span style="color: #1F497D">
+
CAGCTG<u><span style="color: #1F497D">ctgcag</span><span style="color: #B8CCE4">cggccgc</span></u><b>t</b><u><span style="color: #F79646">actagt</span></u><b>atta</b><u><span style="color: #CC00FF">accggt</span></u>GCTCGGGATTTGCAAG
-
ctgcag</span><span color: #B8CCE4">cggccgc</span><span>t<span style="color: #F79646">actagt</span>atta<span style="color: #CC00FF">accggt</span>ATGATGATTCAACCATGGAAC</span></p>
+
<br><br>
<br><br>
-
In order to get a sequence without an internal restriction sites of one of the BioBrick standards the XbaI-restriction site was altered without changing the encoded amino acid(TCT=Serin (TC(T,A,G,C)).
+
In order to get a sequence without an internal restriction sites of one of the BioBrick standards the SpeI-restriction site was altered without changing the encoded amino acid (ACT=Threonine (AC(T,A,G,C)).
<br><br>
<br><br>
-
<b>Primers for Pfu-mutagenese:</b>
+
<b>Primers for Pfu-mutagenese: </b>
<br>
<br>
-
<b>Forward primer (5’->3’) (24 bp)</b>
+
<b>Forward primer (5’->3’): (28 bp)</b>
<br>
<br>
-
GCAAACTCT<span style="background: black">TC</span><span style="background: red">A</span><span style="background: black">AGA</span>GCTAGAGAG
+
GGACAAGACGTT<span style="background: black">AC</span><span style="background: red">G</span><span style="background: black">AGT</span>CAGAAAATCG
<br><br>
<br><br>
-
<b>Reverse primer (5’->3’) (24 bp)</b>
+
<b>Reverse primer (5’->3’): (28 bp)</b>
 +
<br>
 +
CGATTTTCTG<span style="background: black">ACT</span><span style="background: red">C</span><span style="background: black">GT</span>AACGTCTTGTCC
<br>
<br>
-
CTCTCTAGC<span style="background: black">TCT</span><span style="background: red">T</span><span style="background: black">GA</span>AGAGTTTGC
 
-
 
<br>
<br>
</div>
</div>
Line 701: Line 746:
<br>
<br>
<div class="heading">
<div class="heading">
-
<a name="PIF6"></a>
+
<a name="PHYB642"></a>
-
Phytochrome Interacting Factor-6 (PIF6) - <a target="_blank" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K365001"><font color="#E9AF03">BBa_K365001</font></a>  
+
Phytochrome B (aa 1-642) - <a target="_blank" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K365003"><font color="#E9AF03">BBa_K365003</font></a>
</div>
</div>
<div class="desc">
<div class="desc">
Line 708: Line 753:
<br>
<br>
<p><b>Background:</b></p>
<p><b>Background:</b></p>
-
For the design of the first engineered system that achieved to enable the spatiotemporal control of PhyB-PIF interactions in in-vivo experiments, <a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">(Lim & Voigt, 2009.)</a> screened multiple potential  phytochrome–PIF pairs by a fluorescence translocation assay in NIH3T3 cells. They measured the red-light-induced translocation of yellow fluorescent protein (YFP) fused to PIF domains to coexpressed phytochrome domains fused through a flexible linker to mCherry and localized to the plasma membrane by a carboxyterminalpolybasic, prenylation sequence from Kras.  Of all previously reported PIF domains, only the N terminus of PIF6 is strong enough to cause significant translocation of YFP to the membrane.  
+
Phytochromes characterised by a red/far-red photochromicity. Through red-light (650–670 nm) absorption the phytochrome undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of the PIF. This light-sensitive interaction has been mapped to the 650-residue amino-terminal photosensory core of PhyB <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[42]</a></i>. The process is completely reversible through absorption in the near infra-red spectrum (705–740 nm).  
<br><br>
<br><br>
-
<center>
+
The photoreceptor protein PhyB serves for the light-dependent activation of the system, therefore it was fused to the N-teminal of the ClpX-trimer.  
-
<img src="https://static.igem.org/mediawiki/2010/c/c9/ESBS-Strasbourg-Pif6ex.jpg">
+
-
</center>
+
-
<center>
+
-
<i><font size="1">Fig.1: Implied system of Lim and Voigt (2009) to screen potential phytochrome-PIF<br>pairs in a fluorescence translocation assay. </font></i>
+
-
</center>
+
<br><br>
<br><br>
<p><b>Conception:</b></p>
<p><b>Conception:</b></p>
-
We chose used the same sequence of the last 100-residue N-terminal activated phytochrome binding (APB) domain of PIF6, which was already successfully used by <a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">(Lim & Voigt, 2009.)</a>.
+
As mentioned before it has been shown that the PIF-interaction with the PhyB photosensory core (residues 1–650) is irreversible in infrared light in in vivo-application <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[42]</a></i>. Nevertheless, the binding strength and kinetic parameters depend on the composition and nature of the individual system, so we decided to include also this shorter variant of PhyB in our tests.
<br><br>
<br><br>
<center>
<center>
-
<img src="https://static.igem.org/mediawiki/2010/1/13/ESBS-Strasbourg-Gene.png" width="100px" height="42px" border="0">
+
<img src="https://static.igem.org/mediawiki/2010/9/93/ESBS-Strasbourg-PhyB650.png" width="250px" height="167px" border="0">
</center>
</center>
-
<center>
 
-
<i><font size="2">PIF6</font></i>
 
-
</center>
 
-
 
<br><br>
<br><br>
-
The plasmid containing the PIF6-sequence was provided by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Acknowledgment#weber">the laboratory of Wilfried Weber</a></i> from the University of Freiburg. For the synthesis of the Pif6 BioBrick primers containing the sites of the Fusion Protein BioBrick Assembly Standard were used.
+
The plasmid containing the PhyB-sequence was provided by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Acknowledgment#weber">the laboratory of Wilfried Weber</a></i> from the University of Freiburg. To create the BioBrick part the sequence was amplified with primers containing the standard prefix with ATG and the fusion suffix of the Fusion Protein Assembly Standard.
<br><br>
<br><br>
-
<b>Forward primer (5’->3’): 54 bp</b>
+
<b>Forward primer (5’->3’): (42 bp)</b>
<br>
<br>
-
GGATCC<span style="color:red">gaattc</span><span style="color: #B8CCE4">gcggccgc</span>t<span style="color:#00B050">tctaga</span>tg<b><span style="color:#009999">gccggc</span></b>ATGATGTTCTTACCAACCG
+
GGATCC<span style="color:red">gaattc</span><span style="color: #B8CCE4">gcggccgc</span>t<span style="color:#00B050">tctag</span>ATGGTTTCCGGAGTC
<br><br>
<br><br>
-
<b>Reverse primer (5’->3’): 58 bp</b>
+
<b>Reverse primer (5’->3’) : (53 bp)</b>
 +
<br>
 +
CAGCTG<u><span style="color: #1F497D">ctgcag</span><span style="color: #B8CCE4">cggccgc</span></u><b>t</b><u><span style="color: #F79646">actagt</span></u><b>atta</b><u><span style="color: #CC00FF">accggt</span></u>CCCCGCCATATCCCTAC
<br>
<br>
-
CAGCTG<span color: #1F497D">ctgcag</span><span color: #B8CCE4">cggccgc</span><span>t<span style="color: #F79646">actagt</span>atta<span style="color: #CC00FF">accggt</span>GTCAACATGTTTATTGCTTTCC
 
<br>
<br>
</div>
</div>
</td>
</td>
</tr>
</tr>
-
 
-
<tr>
 
-
<td width="750" height="10" bgcolor="#222222">
 
-
</td>
 
-
</tr>
 
-
 
<tr>
<tr>
<td width="750" bgcolor="#414141"">
<td width="750" bgcolor="#414141"">
<br>
<br>
<div class="heading">
<div class="heading">
-
<a name="PHYB908"></a>
+
<a name="PIF3"></a>
-
Phytochrome B (aa 1-908) - <a target="_blank" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K365002"><font color="#E9AF03">BBa_K365002</font></a>
+
Phytochrome Interacting Factor-3 (PIF3) - <a target="_blank" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K365000"><font color="#E9AF03">BBa_K365000</font></a>
</div>
</div>
<div class="desc">
<div class="desc">
Line 758: Line 789:
<br>
<br>
<p><b>Background:</b></p>
<p><b>Background:</b></p>
-
Phytochromes characterised by a red/far-red photochromicity. Through red-light (650–670 nm) absorption the phytochrome undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of the PIF. This light-sensitive interaction has been mapped to the 650-residue amino-terminal photosensory core of PhyB <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">(Khanna et al., 2004)</a></i>. The process is completely reversible through absorption in the near infra-red spectrum (705–740 nm).  
+
PIF3 is a downstream transcription factor in a well studied signaling pathway of A. thaliana, upon stimulation with red (650 nm) light, it binds directly to PhyB and translocates to the nucleus as a heterodimer where it modulates the transcription of response genes. PIF3 binds only the red-light-exposed form of phytochrome, Pfr, and shows no measurable binding affinity for the dark- or infrared-exposed Pr state.<br>
-
<br><br>
+
In our system target proteins are fused to PIF3 and tagged with the DAS degradation sequence which, through light activation, brings the degradation tag in proximity to ClpX.<br>
-
The photoreceptor protein PhyB serves for the light-dependent activation of the system, therefore it will be fused to the N-teminal of the ClpX-trimer.
+
<br>
-
<br><br>
+
<p><b>Conception:</b></p>
<p><b>Conception:</b></p>
-
In in-vivo applications it has been shown that the PIF-interaction with the PhyB photosensory core (residues 1–650) is irreversible in infrared light. <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">Lim & Voigt (2009)</a></i> demonstrated by assaying PIF6 (which has the strongest interactions of all previously reported PIF domains) against different variants of PhyB that the tandem C-terminal PAS domains (residues 1-908)of plant phytochromes are necessary to confer rapid photoreversibility under infrared light.
+
The light-sensitive interaction with PhyB has been mapped to the first 100-residue N-terminal activated phytochrome binding (APB) domain of PIF3 <a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[42].</a><br>
-
The original sequence contains a SpeI restriction within the first 908 residues.
+
We chose this sequence, as it has already been successfully used in different synthetic in vitro applications that benefitted from its light-sensitive interactions with PhyB. The original sequence contains an XbaI restriction site.
<br><br>
<br><br>
<center>
<center>
-
<img src="https://static.igem.org/mediawiki/2010/1/13/ESBS-Strasbourg-Gene.png" width="100px" height="42px" border="0">
+
<img src="https://static.igem.org/mediawiki/2010/8/8b/ESBS-Strasbourg-PIF3.png" width="250px" height="167px" border="0">
</center>
</center>
<center>
<center>
-
<i><font size="2">PhyB 900</font></i>
+
<a><i><b><font color="#FF0000" size="2">There is no PDB structure for the PIF3<br>The stucture below is modeled by homology using the server I-TASSER</font></i></b></a></font>
</center>
</center>
 +
 +
<br>
 +
The plasmid containing the PIF3-sequence was provided by <a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Acknowledgment#kircher">the laboratory of Stephan Kircher</a> from the University of Freiburg. For the synthesis of the BioBrick part primers containing the sites of the Fusion Protein BioBrick Assembly Standard were used.
<br><br>
<br><br>
-
The plasmid containing the PhyB-sequence was provided by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Acknowledgment#weber">the laboratory of Wilfried Weber</a></i> from the University of Freiburg. To create the BioBrick part the sequence was amplified with primers containing the standard prefix with ATG and the fusion suffix of the Fusion Protein Assembly Standard.
+
<b>Forward primer (5’->3’): 51 bp</b>
-
<br><br> 
+
-
<b>Forward primer (5’->3’): (41bp)</b>
+
<br>
<br>
-
GGATCC<span style="color:red">gaattc</span><span style="color: #B8CCE4">gcggccgc</span>t<span style="color:#00B050">tctag</span>ATGGTTTCCGGAGTC
+
<span>
 +
GGATCC<span style="color:red">gaattc</span><span style="color: #B8CCE4">gcggccgc</span>t<span style="color:#00B050">tctaga</span>tg<b><span style="color:#009999">gccggc</span></b>ATGCCTCTGTTTGAGC</span></p>
<br><br>
<br><br>
-
<b>Reverse primer (5’->3’): (52 bp)</b>
+
<b>Reverse primer (5’->3’): 51 bp</b>
<br>
<br>
-
CAGCTG<u><span style="color: #1F497D">ctgcag</span><span style="color: #B8CCE4">cggccgc</span></u><b>t</b><u><span style="color: #F79646">actagt</span></u><b>atta</b><u><span style="color: #CC00FF">accggt</span></u>GCTCGGGATTTGCAAG
+
<span style="color: #1F497D">
 +
ctgcag</span><span color: #B8CCE4">cggccgc</span><span>t<span style="color: #F79646">actagt</span>atta<span style="color: #CC00FF">accggt</span>ATGATGATTCAACCATGGAAC</span></p>
<br><br>
<br><br>
-
In order to get a sequence without an internal restriction sites of one of the BioBrick standards theSpeI-restriction site was altered without changing the encoded amino acid (ACT=Threonine (AC(T,A,G,C)).
+
In order to get a sequence without an internal restriction sites of one of the BioBrick standards the XbaI-restriction site was altered without changing the encoded amino acid(TCT=Serin (TC(T,A,G,C)).
<br><br>
<br><br>
-
<b>Primers for Pfu-mutagenese: </b>
+
<b>Primers for Pfu-mutagenese:</b>
<br>
<br>
-
<b>Forward primer (5’->3’): (28 bp)</b>
+
<b>Forward primer (5’->3’) (24 bp)</b>
<br>
<br>
-
GGACAAGACGTT<span style="background: black">AC</span><span style="background: red">G</span><span style="background: black">AGT</span>CAGAAAATCG
+
GCAAACTCT<span style="background: black">TC</span><span style="background: red">A</span><span style="background: black">AGA</span>GCTAGAGAG
<br><br>
<br><br>
-
<b>Reverse primer (5’->3’): (27 bp)</b>
+
<b>Reverse primer (5’->3’) (24 bp)</b>
-
<br>
+
-
CGATTTTCTG<span style="background: black">ACT</span><span style="background: red">C</span><span style="background: black">GT</span>AACGTCTTGTC
+
<br>
<br>
 +
CTCTCTAGC<span style="background: black">TCT</span><span style="background: red">T</span><span style="background: black">GA</span>AGAGTTTGC
 +
<br>
<br>
</div>
</div>
Line 809: Line 842:
<br>
<br>
<div class="heading">
<div class="heading">
-
<a name="PHYB642"></a>
+
<a name="PIF6"></a>
-
Phytochrome B (aa 1-642) - <a target="_blank" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K365003"><font color="#E9AF03">BBa_K365003</font></a>
+
Phytochrome Interacting Factor-6 (PIF6) - <a target="_blank" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K365001"><font color="#E9AF03">BBa_K365001</font></a>  
</div>
</div>
<div class="desc">
<div class="desc">
Line 816: Line 849:
<br>
<br>
<p><b>Background:</b></p>
<p><b>Background:</b></p>
-
Phytochromes characterised by a red/far-red photochromicity. Through red-light (650–670 nm) absorption the phytochrome undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of the PIF. This light-sensitive interaction has been mapped to the 650-residue amino-terminal photosensory core of PhyB <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">(Lim & Voigt 2009)</a></i>. The process is completely reversible through absorption in the near infra-red spectrum (705–740 nm).  
+
For the design of the first engineered system that achieved to enable the spatiotemporal control of PhyB-PIF interactions in in-vivo experiments, <a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[42].</a> screened multiple potential  phytochrome–PIF pairs by a fluorescence translocation assay in NIH3T3 cells. They measured the red-light-induced translocation of yellow fluorescent protein (YFP) fused to PIF domains to co-expressed phytochrome domains fused through a flexible linker to mCherry and localized to the plasma membrane by a carboxyterminalpolybasic, prenylation sequence from Kras.  Of all previously reported PIF domains, only the N terminus of PIF6 is strong enough to cause significant translocation of YFP to the membrane.  
<br><br>
<br><br>
-
The photoreceptor protein PhyB serves for the light-dependent activation of the system, therefore it was fused to the N-teminal of the ClpX-trimer.  
+
<center>
 +
<img src="https://static.igem.org/mediawiki/2010/c/c9/ESBS-Strasbourg-Pif6ex.jpg">
 +
</center>
 +
<center>
 +
<i><font size="1">Fig.1: Implied system of Lim and Voigt (2009) to screen potential phytochrome-PIF<br>pairs in a fluorescence translocation assay. </font></i>
 +
</center>
<br><br>
<br><br>
<p><b>Conception:</b></p>
<p><b>Conception:</b></p>
-
As mentioned before it has been shown that the PIF-interaction with the PhyB photosensory core (residues 1–650) is irreversible in infrared light in in vivo-application <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">(Lim & Voigt 2009)</a></i>. Nevertheless, the binding strength and kinetic parameters depend on the composition and nature of the individual system, so we decided to include also this shorter variant of PhyB in our tests.
+
We chose used the same sequence of the last 100-residue N-terminal activated phytochrome binding (APB) domain of PIF6, which was already successfully used by <a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[42].</a>.
<br><br>
<br><br>
<center>
<center>
-
<img src="https://static.igem.org/mediawiki/2010/1/13/ESBS-Strasbourg-Gene.png" width="100px" height="42px" border="0">
+
<img src="https://static.igem.org/mediawiki/2010/2/23/ESBS-Strasbourg-PIF6.png" width="250px" height="167px" border="0">
</center>
</center>
<center>
<center>
-
<i><font size="2">PhyB 650</font></i>
+
<a><i><b><font color="#FF0000" size="2">There is no PDB structure for the PIF6<br>The stucture below is modeled by homology using the server I-TASSER</font></i></b></a></font>
</center>
</center>
 +
 +
<br><br>
<br><br>
-
The plasmid containing the PhyB-sequence was provided by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Acknowledgment#weber">the laboratory of Wilfried Weber</a></i> from the University of Freiburg. To create the BioBrick part the sequence was amplified with primers containing the standard prefix with ATG and the fusion suffix of the Fusion Protein Assembly Standard.
+
The plasmid containing the PIF6-sequence was provided by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Acknowledgment#weber">the laboratory of Wilfried Weber</a></i> from the University of Freiburg. For the synthesis of the Pif6 BioBrick primers containing the sites of the Fusion Protein BioBrick Assembly Standard were used.
<br><br>
<br><br>
-
<b>Forward primer (5’->3’): (42 bp)</b>
+
<b>Forward primer (5’->3’): 54 bp</b>
<br>
<br>
-
GGATCC<span style="color:red">gaattc</span><span style="color: #B8CCE4">gcggccgc</span>t<span style="color:#00B050">tctag</span>ATGGTTTCCGGAGTC
+
GGATCC<span style="color:red">gaattc</span><span style="color: #B8CCE4">gcggccgc</span>t<span style="color:#00B050">tctaga</span>tg<b><span style="color:#009999">gccggc</span></b>ATGATGTTCTTACCAACCG
<br><br>
<br><br>
-
<b>Reverse primer (5’->3’) : (53 bp)</b>
+
<b>Reverse primer (5’->3’): 58 bp</b>
-
<br>
+
-
CAGCTG<u><span style="color: #1F497D">ctgcag</span><span style="color: #B8CCE4">cggccgc</span></u><b>t</b><u><span style="color: #F79646">actagt</span></u><b>atta</b><u><span style="color: #CC00FF">accggt</span></u>CCCCGCCATATCCCTAC
+
<br>
<br>
 +
CAGCTG<span color: #1F497D">ctgcag</span><span color: #B8CCE4">cggccgc</span><span>t<span style="color: #F79646">actagt</span>atta<span style="color: #CC00FF">accggt</span>GTCAACATGTTTATTGCTTTCC
<br>
<br>
</div>
</div>
</td>
</td>
</tr>
</tr>
 +
 +
<tr>
 +
<td width="750" height="10" bgcolor="#222222">
 +
</td>
 +
</tr>
 +
 +
<tr>
<tr>
Line 875: Line 921:
<br>
<br>
<center>
<center>
-
<img src="https://static.igem.org/mediawiki/2010/1/13/ESBS-Strasbourg-Gene.png" width="100px" height="42px" border="0">
+
<img src="https://static.igem.org/mediawiki/2010/3/32/ESBS-Strasbourg-(-N)ClpX.png" width="250px" height="167px" border="0">
-
</center>
+
-
<center>
+
-
<i><font size="2">∆N-ClpX</font></i>
+
</center>
</center>
<br><br>
<br><br>
-
<b>Problem:<span style="background: black">2 AgeI and 2 EcoRI sides</b>
+
<b>Problem: <span style="background: black">2 AgeI and 2 EcoRI sides</b>
<br><br>
<br><br>
<b>Primers for cloning ClpX out of the E.Coli genome.</b>
<b>Primers for cloning ClpX out of the E.Coli genome.</b>
Line 896: Line 939:
<br><br>
<br><br>
-
<b>Primers for Pfu-mutagenese<b/>
+
<b>Primers for Pfu-mutagenese</b>
-
<p>1. ACC=Thr AC(A,T,G,C), GGT=Gly GG(A,T,G,C)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AgeI site</p>
+
<p><b>1. ACC=Thr AC(A,T,G,C), GGT=Gly GG(A,T,G,C)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AgeI site</b></p>
<br>
<br>
<b>Forward primer (5’->3’) (31 bp)</b>
<b>Forward primer (5’->3’) (31 bp)</b>
Line 903: Line 946:
CTGATCGGTCCG<span style="background: black">AC</span><span style="background: red">T</span><span style="background: Black">GGT</span>TCCGGTAAAACGC
CTGATCGGTCCG<span style="background: black">AC</span><span style="background: red">T</span><span style="background: Black">GGT</span>TCCGGTAAAACGC
<br><br>
<br><br>
-
<b>Reverse primer (5’->3’) (29 bp)</b>
+
<b>Reverse primer (5’->3’) (31 bp)</b>
<br>
<br>
GCGTTTTACCGGA<span style="background: black">ACC</span><span style="background: red">A</span><span style="background: black">GT</span>CGGACCGATCAG<span style="color:red"></span>
GCGTTTTACCGGA<span style="background: black">ACC</span><span style="background: red">A</span><span style="background: black">GT</span>CGGACCGATCAG<span style="color:red"></span>
<br><br>
<br><br>
-
<p>2. GAA=Glu GA(A,G), TTC=Phe TT(C,T)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;EcoRI site</p>
+
<p><b>2. GAA=Glu GA(A,G), TTC=Phe TT(C,T)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;EcoRI site</b></p>
<br>
<br>
<b>Forward primer (5’->3’) (28 bp)</b>
<b>Forward primer (5’->3’) (28 bp)</b>
Line 919: Line 962:
<br><br>
<br><br>
-
<p>3. GAA=Glu GA(A,G), TTC=Phe TT(C,T)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;EcoRI site</p>
+
<p><b>3. GAA=Glu GA(A,G), TTC=Phe TT(C,T)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;EcoRI site</b></p>
<br>
<br>
<b>Forward primer (5’->3’) (25 bp)</b>
<b>Forward primer (5’->3’) (25 bp)</b>
Line 930: Line 973:
<br><br>
<br><br>
-
<p>4. ACC=Thr AC(A,T,G,C), GGT=Gly GG(A,T,G,C)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AgeI site
+
<p><b>4. ACC=Thr AC(A,T,G,C), GGT=Gly GG(A,T,G,C)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AgeI site</b></p>
<br>
<br>
<b>Forward primer (5’->3’) (24 bp)</b>
<b>Forward primer (5’->3’) (24 bp)</b>
Line 946: Line 989:
<b>Forward primer (5’->3’):</b>
<b>Forward primer (5’->3’):</b>
<br>
<br>
-
NNNNNN<span style="color:red">gaattc</span><span style="color: #B8CCE4">gcggccgc</span>t<span style="color:#00B050">tctaga</span>tg<b><span style="color:#009999">gccggc</span></b></span><span>CGCAGT</span><span>GCGCTACCGACGCCGC
+
GGATCC<span style="color:red">gaattc</span><span style="color: #B8CCE4">gcggccgc</span>t<span style="color:#00B050">tctaga</span>tg<b><span style="color:#009999">gccggc</span></b></span><span>CGCAGT</span><span>GCGCTACCGACGCCGC
<br><br>
<br><br>
<b>Reverse primer (5’->3’):</b>
<b>Reverse primer (5’->3’):</b>
<br>
<br>
-
NNNNNN<span style="color: #1F497D">ctgcag</span><span style="color: #B8CCE4">cggccgc</span>t<span style="color: #F79646">actagt</span>atta<span style="color: #CC00FF">accggt</span>TTCACCAGATGCCTGTTGCGC
+
CAGCTG<span style="color: #1F497D">ctgcag</span><span style="color: #B8CCE4">cggccgc</span>t<span style="color: #F79646">actagt</span>atta<span style="color: #CC00FF">accggt</span>TTCACCAGATGCCTGTTGCGC
<br>
<br>
Line 977: Line 1,020:
<br><br>
<br><br>
<p><b>Conception:</b></p>
<p><b>Conception:</b></p>
-
We chose to use the same linker, which was already successfully used by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">Baker and Sauer (2009)</a></i> to construct the ClpX trimer. It is a twenty amino acid linker (ASGAGGSEGGGSEGGTSGAT). The codon usage of E. coli <i><a href="http://www.geneinfinity.org/sp_codonusage.html">(http://www.geneinfinity.org/sp_codonusage.html)</a></i> has been used to decide the DNA sequence, in addition RFC 25 fusion prefix and suffix have been added to the sequence.
+
We chose to use the same linker, which was already successfully used by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[2]</a></i> to construct the ClpX trimer. It is a twenty amino acid linker (ASGAGGSEGGGSEGGTSGAT). The codon usage of E. coli <i><a href="http://www.geneinfinity.org/sp_codonusage.html">(http://www.geneinfinity.org/sp_codonusage.html)</a></i> has been used to decide the DNA sequence, in addition RFC 25 fusion prefix and suffix have been added to the sequence.
<br><br>
<br><br>
<center>
<center>
-
<img src="https://static.igem.org/mediawiki/2010/1/13/ESBS-Strasbourg-Gene.png" width="100px" height="42px" border="0">
+
<img src="https://static.igem.org/mediawiki/2010/d/d1/ESBS-Strasbourg-linker.png" width="250px" height="167px" border="0">
-
</center>
+
-
<center>
+
-
<i><font size="2">Linker</font></i>
+
</center>
</center>
Line 1,043: Line 1,083:
<br>
<br>
<p><b>Background:</b></p>
<p><b>Background:</b></p>
-
LAA tag is a C-terminal region of the natural ssrA-recognition sequence of E. Coli that interacts with the ClpXP protease. A protein fused with this tag will be preferentially degraded by the ClpX protease without need of an adaptor protein <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">Baker and Sauer (2006)</a></i>. This tag serves as positive control for the functionality of the composed ClpXP and the PhyB-ClpXP fusion protein.
+
LAA tag is a C-terminal region of the natural ssrA-recognition sequence of E. Coli that interacts with the ClpXP protease. A protein fused with this tag will be preferentially degraded by the ClpX protease without need of an adaptor protein <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[6]</a></i>. This tag serves as positive control for the functionality of the composed ClpXP and the PhyB-ClpXP fusion protein.
<br><br>
<br><br>
<p><b>Conception:</b></p>
<p><b>Conception:</b></p>
-
We chose to use the same tag, which was already successfully used by used by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">Baker and Sauer (2006)</a></i>. It is a eleven amino acid tag (AANDENYALAA). At the end of the coding sequence a double stop codon was added (<span style="color: #F79646">ACTAGT</span>). The codon usage of E. coli <i><a href="http://www.geneinfinity.org/sp_codonusage.html">(http://www.geneinfinity.org/sp_codonusage.html)</a></i> has been used to decide the DNA sequence and NgoMIV and Pst1 restriction sites have been added to the sequence.
+
We chose to use the same tag, which was already successfully used by used by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[6]</a></i>. It is a eleven amino acid tag (AANDENYALAA). At the end of the coding sequence a double stop codon was added (<span style="color: #F79646">ACTAGT</span>). The codon usage of E. coli <i><a href="http://www.geneinfinity.org/sp_codonusage.html">(http://www.geneinfinity.org/sp_codonusage.html)</a></i> has been used to decide the DNA sequence and NgoMIV and Pst1 restriction sites have been added to the sequence.
<br><br>
<br><br>
-
The linker has been order as two separate, <span style="color: #009999">NgoMIV </span>+ <span style="color: blue">Pst1</span> precut primers, which were hybridized in order to obtain the complete tag sequence.
+
The LAA-tag has been order as two separate, <span style="color: #009999">NgoMIV </span>+ <span style="color: blue">Pst1</span> precut primers, which were hybridized in order to obtain the degradation sequence.
<br><br>
<br><br>
<center>
<center>
-
<img src="https://static.igem.org/mediawiki/2010/1/13/ESBS-Strasbourg-Gene.png" width="100px" height="42px" border="0">
+
<img src="https://static.igem.org/mediawiki/2010/3/3e/ESBS-Strasbourg-LAAtag.png" width="250px" height="167px" border="0">
</center>
</center>
<center>
<center>
Line 1,089: Line 1,129:
<br>
<br>
<p><b>Background:</b></p>
<p><b>Background:</b></p>
-
The DAS tag presents a C-terminal recognition  sequence that has been artificially altered so that it has weakened interactions with ClpXP and depends on an adaptor <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">Baker and Sauer (2006)</a></i>. In E. coli, the adaptor SspB tethers specifically tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. In our system, the role of the adaptor-protein SspB has been assumed by Pif3/6. So only light-induced activation can lead to binding and efficient degradation of DAS+4 bearing constructs.  
+
The DAS tag presents a C-terminal recognition  sequence that has been artificially altered so that it has weakened interactions with ClpXP and depends on an adaptor <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[6]</a></i>. In E. coli, the adaptor SspB tethers specifically tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. In our system, the role of the adaptor-protein SspB has been assumed by Pif3/6. So only light-induced activation can lead to binding and efficient degradation of DAS bearing constructs.  
<br><br>
<br><br>
<p><b>Conception:</b></p>
<p><b>Conception:</b></p>
-
We decided to use the same tag, which was already successfully used by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">Baker and Sauer (2006)</a></i>. It is a eleven amino acid tag (AANDENYADAS). At the end of the coding sequence a double stop codon was added (<span style="color: #F79646">ACTAGT</span>). The codon usage of E. coli <i><a href="http://www.geneinfinity.org/sp_codonusage.html">(http://www.geneinfinity.org/sp_codonusage.html)</a></i> has been used to decide the DNA sequence, in addition NgoMIV and PST1 restriction sites have been added to the sequence.
+
We decided to use the same tag, which was already successfully used by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[6]</a></i>. It is a eleven amino acid tag (AANDENYADAS). At the end of the coding sequence a double stop codon was added (<span style="color: #F79646">ACTAGT</span>). The codon usage of E. coli <i><a href="http://www.geneinfinity.org/sp_codonusage.html">(http://www.geneinfinity.org/sp_codonusage.html)</a></i> has been used to decide the DNA sequence, in addition NgoMIV and PST1 restriction sites have been added to the sequence.
<br><br>
<br><br>
-
The linker has been order as two separate, <span style="color: #009999">NgoMIV </span>+ <span style="color: blue">Pst1</span> precut primers, which were hybridized in order to obtain the complete linker sequence.
+
The DAS-tag has been order as two separate, <span style="color: #009999">NgoMIV </span>+ <span style="color: blue">Pst1</span> precut primers, which were hybridized in order to obtain the degradation sequence.
<br><br>
<br><br>
<center>
<center>
-
<img src="https://static.igem.org/mediawiki/2010/1/13/ESBS-Strasbourg-Gene.png" width="100px" height="42px" border="0">
+
<img src="https://static.igem.org/mediawiki/2010/2/23/ESBS-Strasbourg-DAStag.png" width="250px" height="167px" border="0">
-
</center>
+
-
<center>
+
-
<i><font size="2">DAS Tag</font></i>
+
</center>
</center>
<br><br>
<br><br>
Line 1,140: Line 1,177:
The λO- tag is the N-terminal equalent to the DAS tag. Degradation of proteins bearing the N-terminal λO- tag normally requires the N-domain of ClpX, which is missing in the PhyB-linker-[ClpX]3 variant.  
The λO- tag is the N-terminal equalent to the DAS tag. Degradation of proteins bearing the N-terminal λO- tag normally requires the N-domain of ClpX, which is missing in the PhyB-linker-[ClpX]3 variant.  
<br><br>
<br><br>
-
<i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">Baker and Sauer (2009)</a></i> used this tag to test an artificial tethering system and demonstrated that it can serve as degradation signal for substrates that are tethered to ClpX.
+
<i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[2]</a></i> used this tag to test an artificial tethering system and demonstrated that it can serve as degradation signal for substrates that are tethered to ClpX.
<br><br>
<br><br>
<p><b>Conception:</b></p>
<p><b>Conception:</b></p>
-
We chose to use the same sequence, which was already successfully used by used by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">Baker and Sauer (2009)</a></i>: NH2-TNTAKILNFGR. The codon usage of E. coli <i><a href="http://www.geneinfinity.org/sp_codonusage.html">(http://www.geneinfinity.org/sp_codonusage.html)</a></i> has been used to decide the DNA sequence, in addition NgoMIV and AgeI restriction sites have been added to the sequence.  
+
We chose to use the same sequence, which was already successfully used by used by <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[2]</a></i>: NH2-TNTAKILNFGR. The codon usage of E. coli <i><a href="http://www.geneinfinity.org/sp_codonusage.html">(http://www.geneinfinity.org/sp_codonusage.html)</a></i> has been used to decide the DNA sequence, in addition NgoMIV and AgeI restriction sites have been added to the sequence.  
<br><br>
<br><br>
-
The linker has been order as two separate, <span style="color: #009999">NgoMIV </span>+ <span style="color: blue">Pst1</span> precut primers, which were hybridized in order to obtain the complete linker sequence.
+
The tag has been order as two separate, <span style="color: #009999">NgoMIV </span>+ <span style="color: blue">Pst1</span> precut primers, which were hybridized in order to obtain the whole degradation tag.
<br><br>
<br><br>
<center>
<center>
-
<img src="https://static.igem.org/mediawiki/2010/1/13/ESBS-Strasbourg-Gene.png" width="100px" height="42px" border="0">
+
<img src="https://static.igem.org/mediawiki/2010/0/0a/ESBS-Strasbourg-Lambdatag.png" width="250px" height="167px" border="0">
-
</center>
+
-
<center>
+
-
<i><font size="2">Lambda Tag</font></i>
+
</center>
</center>
<br><br>
<br><br>
Line 1,180: Line 1,214:
<br>
<br>
<br>
<br>
-
...
+
<p><b>Background</b></p>
 +
The GFP super fold protein originated from the BBa_I746915. This old biobrick contains the normal standard prefix and suffix. When this GFP is now fused to another biobrick a stop codon is created which would terminate the transcription of the fused biobrick. To avoid this inconvenience, we improved this biobrick to the fusion standard BBF RFC 25. This new biobrick can now be added to any biobrick with compatible restriction sides without creating a stop codon.
 +
<br><br>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2010/c/cd/ESBS-Strasbourg-GFP.png" width="300px" height="163px" border="0">
 +
</center>
 +
<br>
 +
<p><b>Conception</b></p>
 +
Standard PCR techniques were used. The Primer for amplification contained the fusion pre- and suffix and compatible base pair for the GFP gene.
 +
 
<br>
<br>
</div>
</div>
Line 1,201: Line 1,244:
<br>
<br>
<br>
<br>
 +
<p><b>Background:</b></p>
 +
This final construct will be the inducible part of the protease system. This permits the linking of the phytochrome B to the Clpx trimer which forms the light-dependent activation part of our degradation system.
 +
<br><br>
<center>
<center>
-
<img src="https://static.igem.org/mediawiki/2010/9/94/ESBS-Strasbourg-Phyb-clpx.png" width="296px" height="206px" border="0">
+
<img src="https://static.igem.org/mediawiki/2010/2/2d/ESBS-Strasbourg-PhyB650-clpx3.png" width="296px" height="206px" border="0">
</center>
</center>
<br>
<br>
-
<center>
 
-
<img src="https://static.igem.org/mediawiki/2010/f/fe/ESBS-Strasbourg-PhyB642-clpx3.jpg" width="648px" height="56px" border="0">
 
-
</center>
 
-
<br>
 
-
...
 
<br>
<br>
 +
<p><b>Conception:</b></p>
 +
The standards cloning methods were used. This includes digestion of the vector containing linker- ClpX trimer and the PhyB part then the ligation of them and to finish the transformation of competente cells with this plasmid.
 +
<br><br>
</div>
</div>
</td>
</td>
Line 1,230: Line 1,274:
<br>
<br>
<br>
<br>
-
...
+
<p><b>Background:</b></p>
 +
This final construct will be the inducible part of the protease system. This permits the linking of the phytochrome B to the Clpx trimer which forms the light-dependent activation part of our degradation system.
 +
<br><br>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2010/0/0f/ESBS-Strasbourg-PhyB908-clpx3.png" width="296px" height="206px" border="0">
 +
</center>
 +
<br>
 +
<br>
 +
<p><b>Conception:</b></p>
 +
The standards cloning methods were used. This includes digestion of the vector containing linker- ClpX trimer and the PhyB part then the ligation of them and to finish the transformation of competente cells with this plasmid.
 +
<br>
<br>
<br>
</div>
</div>
Line 1,251: Line 1,305:
<br>
<br>
<br>
<br>
-
...
+
<p><b>Background</b></p>
 +
ClpXP is a part of an E.coli protease which consists of three parts, the hexametric ClpX and two heptametrical ClpP subunits. ClpX consists of six identical subunits, each 1092bp long. ClpX recognizes and unfolds protein containing certain tags like LAA and leading them into the catalytic center of this protein complex, the two ClpP units. ClpX has two internal restriction sides for EcoRI and two restriction sides for AgeI.
 +
<br><br>
 +
<p><b>Conception</b></p>
 +
ClpX has two internal restriction sides for EcoRI and two restriction sides for AgeI. The purpose of this first experimental part was to extract the ClpX gene out of the E.coli genome, to alter the internal EcoRI and AgeI sides in the ClpX gene and to fuse iGEM fusion pre- and suffixes to the ClpX sequence in order to get an iGEM Biobrick with standard prefix and suffix standard without internal EcoRI, Not, XbaI, AgeI, SpeI and PstI sides.
 +
<br><br>
 +
The sequence was obtained from the following database for DH5α E.coli cells:
 +
<br>
 +
<p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/gene/945083">
 +
1- http://www.ncbi.nlm.nih.gov/gene/945083</a></span></p>
 +
<p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=456650&to=457924&report=gbwithparts">
 +
2- http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=456650&amp;to=457924&amp;report=gbwithparts</a></p>
 +
 
 +
<br>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2010/d/d1/ESBS-Strasbourg-ClpXfull.png" width="250px" height="167px" border="0">
 +
</center>
 +
<br><br>
 +
<b>Problem:  <span style="background: black">2 AgeI and 2 EcoRI sides</b>
 +
<br><br>
 +
<b>Primers for cloning ClpX out of the E.Coli genome.</b>
 +
<br>
 +
These primes were used to amplificate ClpX from the E.coli genome.
 +
<br><br>
 +
<b>Forward primer (5’->3’) : 31bp</b>
 +
<br>
 +
<u>CGCAGTGCGCTACCGACGCCGCATGAAATTC</u>
 +
<br><br>
 +
<b>Reverse primer (5’->3’) : 32bp</b>
 +
<br>
 +
<u>TTCACCAGATGCCTGTTGCGCTTCCGGCTTGC</u>
 +
<br><br>
 +
 
 +
<b>Primers for Pfu-mutagenese</b>
 +
<p><b>1. ACC=Thr AC(A,T,G,C), GGT=Gly GG(A,T,G,C)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AgeI site</b></p>
 +
<br>
 +
<b>Forward primer (5’->3’) (31 bp)</b>
 +
<br>
 +
CTGATCGGTCCG<span style="background: black">AC</span><span style="background: red">T</span><span style="background: Black">GGT</span>TCCGGTAAAACGC
 +
<br><br>
 +
<b>Reverse primer (5’->3’) (31 bp)</b>
 +
<br>
 +
GCGTTTTACCGGA<span style="background: black">ACC</span><span style="background: red">A</span><span style="background: black">GT</span>CGGACCGATCAG<span style="color:red"></span>
 +
<br><br>
 +
 
 +
<p><b>2. GAA=Glu GA(A,G), TTC=Phe TT(C,T)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;EcoRI site</b></p>
 +
<br>
 +
<b>Forward primer (5’->3’) (28 bp)</b>
 +
<br>
 +
CATCCGCAGCAG<span style="background: black">GA</span><span style="background: red">G</span><span style="background: black">TTC</span>TTGCAGGTTG
 +
<br><br>
 +
<b>Reverse primer (5’->3’) (28 bp)</b>
 +
<br>
 +
CAACCTGCAA<span style="background: black">GAA</span><span style="background: red">C</span><span style="background: black">TC</span>CTGCTGCGGATG
 +
<br><br>
 +
 
 +
<p><b>3. GAA=Glu GA(A,G), TTC=Phe TT(C,T)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;EcoRI site</b></p>
 +
<br>
 +
<b>Forward primer (5’->3’) (25 bp)</b>
 +
<br>
 +
CGTGGATCTG<span style="background: black">GA</span><span style="background: red">G</span><span style="background: black">TTC</span>CGTGACGAG
 +
<br><br>
 +
<b>Reverse primer (5’->3’) (25 bp)</b>
 +
<br>
 +
CTCGTCACG<span style="background: black">GAA</span><span style="background: red">C</span><span style="background: black">TC</span>CAGATCCACG
 +
<br><br>
 +
 
 +
<p><b>4. ACC=Thr AC(A,T,G,C), GGT=Gly GG(A,T,G,C)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AgeI site</b></p>
 +
<br>
 +
<b>Forward primer (5’->3’) (24 bp)</b>
 +
<br>
 +
GGCGCGTAAA<span style="background: black">AC</span><span style="background: red">T</span><span style="background: black">GGT</span>GCCCGTGG
 +
<br><br>
 +
<b>Reverse primer (5’->3’) (24 bp)</b>
 +
<br>
 +
CCACGGGC<span style="background: black">ACC</span><span style="background: red">A</span><span style="background: black">GT</span>TTTACGCGCC
 +
<br><br>
<br>
<br>
</div>
</div>
Line 1,272: Line 1,402:
<br>
<br>
<br>
<br>
-
...
+
<p><b>Background:</b></p>
 +
Phytochrome B needs to be fixed to the Clpx hexametric part of the protease. It is possible to link one phytochrome B per ClpX monomer but this could lead to steric problems. So the decision was made to follow the idea of the publication of Tanja Baker 2009. This permits to have only one phytochrome B for three ClpX units. Moreover the publication proved that the speed of the assembly of two ClpX trimers is quite the same than with ClpX monomers. So the Clpx hexameric part is composed of two ClpX trimers each one couple with a phytochrome B.
 +
<br><br>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2010/3/3f/ESBS-Strasbourg-ClpX-Trimere.png" width="250px" height="132px" border="0">
 +
</center>
 +
<br><br>
 +
<p><b>Conception:</b</p>
 +
ClpX primer was constructed due to cloning methods. The new approach in this method is that the primer was built from a PCR product. The construction of the ClpX dimer was conducted with standard methods. Then in a PCR reaction the ClpX primers located inside of the dimer were used to construct this new primer. The product of this PCR was used as primer for constructing the ClpX trimer.
 +
<br><br>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
<br>
<br>
</div>
</div>
Line 1,293: Line 1,439:
<br>
<br>
<br>
<br>
-
...
+
<p><b>Background:</b></p>
-
<br>
+
This construct was the final step for the ClpX hexamer creation. The two trimers will assemble to the full ClpX unit. Then the phytochrome B was cloned to this trimer. It will also be possible to add any other activation system like hormone receptors to this biobrick instead of phytochrome B, giving this part a huge potential of further applications.
-
</div>
+
<br><br>
-
</td>
+
<center>
-
</tr>
+
<img src="https://static.igem.org/mediawiki/2010/0/06/ESBS-Strasbourg-Linker-ClpX-Trimere.png" width="250px" height="132px" border="0">
 +
</center>
 +
<br><br>
 +
<p><b>Conception:</b></p>
 +
The standards cloning methods were used. This includes digestion of the vector containing ClpX trimer and the linker part then the ligation of them and to finish the transformation of competente cells with this plasmid.
 +
<br><br>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
-
<tr>
 
-
<td width="750" height="10" bgcolor="#222222">
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td width="750" bgcolor="#414141"">
 
-
<br>
 
-
<div class="heading">
 
-
16
 
-
</div>
 
-
<div class="desc">
 
-
<br>
 
-
<br>
 
-
...
 
<br>
<br>
</div>
</div>
</td>
</td>
</tr>
</tr>
-
 
-
 

Latest revision as of 21:47, 27 October 2010

{|

ESBS - Strasbourg


Colored restriction sites :
EcoRI NotI XbaI NgoMIV
AgeI SpeI NotI PstI

Biobricks

  
Let me guide you
Parts Submitted to Registry


      

Part Number: Part Name: Plasmid/Resistance Status
BBa_K365000 Phytochrome Interacting Factor-3 (PIF3) pSB1C3 / Chloramphenicol Sequenced
BBa_K365001 Phytochrome Interacting Factor-6 (PIF6) pSB1C3 / Chloramphenicol Sequenced
BBa_K365002 Phytochrome B (aa 1-908) pSB1C3 / Chloramphenicol Sequenced
BBa_K365003 Phytochrome B (aa 1-642) pSB1C3 / Chloramphenicol Sequenced
BBa_K365004 ∆N-ClpX (aa 61-425) pSB1C3 / Chloramphenicol Sequenced
BBa_K365005 Linker (aa 20) pSB1C3 / Chloramphenicol Sequenced
BBa_K365006 LAA tag pSB1C3 / Chloramphenicol Sequenced
BBa_K365007 DAS tag pSB1C3 / Chloramphenicol Sequenced
BBa_K365008 Lambda tag pSB1C3 / Chloramphenicol Sequenced
BBa_K365009 GFP (super fold) pSB1C3 / Chloramphenicol Sequenced
BBa_K365010 PhyB642-(linker-∆NClpX)3 pSB1C3 / Chloramphenicol Assembled
BBa_K365011 PhyB908-(linker-∆NClpX)3 pSB1C3 / Chloramphenicol Assembled
BBa_K365012 Full-length ClpX pSB1C3 / Chloramphenicol Sequenced
BBa_K365013 ∆NClpX-linker-∆NClpX-linker-∆NClpX pSB1C3 / Chloramphenicol Assembled
BBa_K365014 (linker-∆NClpX)3 pSB1C3 / Chloramphenicol Assembled


Phytochrome B (aa 1-908) - BBa_K365002


Background:

Phytochromes characterised by a red/far-red photochromicity. Through red-light (650–670 nm) absorption the phytochrome undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of the PIF. This light-sensitive interaction has been mapped to the 650-residue amino-terminal photosensory core of PhyB [30]. The process is completely reversible through absorption in the near infra-red spectrum (705–740 nm).

The photoreceptor protein PhyB serves for the light-dependent activation of the system, therefore it will be fused to the N-teminal of the ClpX-trimer.

Conception:

In in-vivo applications it has been shown that the PIF-interaction with the PhyB photosensory core (residues 1–650) is irreversible in infrared light. [42] demonstrated by assaying PIF6 (which has the strongest interactions of all previously reported PIF domains) against different variants of PhyB that the tandem C-terminal PAS domains (residues 1-908)of plant phytochromes are necessary to confer rapid photoreversibility under infrared light. The original sequence contains a SpeI restriction within the first 908 residues.



The plasmid containing the PhyB-sequence was provided by the laboratory of Wilfried Weber from the University of Freiburg. To create the BioBrick part the sequence was amplified with primers containing the standard prefix with ATG and the fusion suffix of the Fusion Protein Assembly Standard.

Forward primer (5’->3’): (41bp)
GGATCCgaattcgcggccgcttctagATGGTTTCCGGAGTC

Reverse primer (5’->3’): (52 bp)
CAGCTGctgcagcggccgctactagtattaaccggtGCTCGGGATTTGCAAG

In order to get a sequence without an internal restriction sites of one of the BioBrick standards the SpeI-restriction site was altered without changing the encoded amino acid (ACT=Threonine (AC(T,A,G,C)).

Primers for Pfu-mutagenese:
Forward primer (5’->3’): (28 bp)
GGACAAGACGTTACGAGTCAGAAAATCG

Reverse primer (5’->3’): (28 bp)
CGATTTTCTGACTCGTAACGTCTTGTCC


Phytochrome B (aa 1-642) - BBa_K365003


Background:

Phytochromes characterised by a red/far-red photochromicity. Through red-light (650–670 nm) absorption the phytochrome undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of the PIF. This light-sensitive interaction has been mapped to the 650-residue amino-terminal photosensory core of PhyB [42]. The process is completely reversible through absorption in the near infra-red spectrum (705–740 nm).

The photoreceptor protein PhyB serves for the light-dependent activation of the system, therefore it was fused to the N-teminal of the ClpX-trimer.

Conception:

As mentioned before it has been shown that the PIF-interaction with the PhyB photosensory core (residues 1–650) is irreversible in infrared light in in vivo-application [42]. Nevertheless, the binding strength and kinetic parameters depend on the composition and nature of the individual system, so we decided to include also this shorter variant of PhyB in our tests.



The plasmid containing the PhyB-sequence was provided by the laboratory of Wilfried Weber from the University of Freiburg. To create the BioBrick part the sequence was amplified with primers containing the standard prefix with ATG and the fusion suffix of the Fusion Protein Assembly Standard.

Forward primer (5’->3’): (42 bp)
GGATCCgaattcgcggccgcttctagATGGTTTCCGGAGTC

Reverse primer (5’->3’) : (53 bp)
CAGCTGctgcagcggccgctactagtattaaccggtCCCCGCCATATCCCTAC


Phytochrome Interacting Factor-3 (PIF3) - BBa_K365000


Background:

PIF3 is a downstream transcription factor in a well studied signaling pathway of A. thaliana, upon stimulation with red (650 nm) light, it binds directly to PhyB and translocates to the nucleus as a heterodimer where it modulates the transcription of response genes. PIF3 binds only the red-light-exposed form of phytochrome, Pfr, and shows no measurable binding affinity for the dark- or infrared-exposed Pr state.
In our system target proteins are fused to PIF3 and tagged with the DAS degradation sequence which, through light activation, brings the degradation tag in proximity to ClpX.

Conception:

The light-sensitive interaction with PhyB has been mapped to the first 100-residue N-terminal activated phytochrome binding (APB) domain of PIF3 [42].
We chose this sequence, as it has already been successfully used in different synthetic in vitro applications that benefitted from its light-sensitive interactions with PhyB. The original sequence contains an XbaI restriction site.

There is no PDB structure for the PIF3
The stucture below is modeled by homology using the server I-TASSER

The plasmid containing the PIF3-sequence was provided by the laboratory of Stephan Kircher from the University of Freiburg. For the synthesis of the BioBrick part primers containing the sites of the Fusion Protein BioBrick Assembly Standard were used.

Forward primer (5’->3’): 51 bp
GGATCCgaattcgcggccgcttctagatggccggcATGCCTCTGTTTGAGC



Reverse primer (5’->3’): 51 bp
ctgcagcggccgctactagtattaaccggtATGATGATTCAACCATGGAAC



In order to get a sequence without an internal restriction sites of one of the BioBrick standards the XbaI-restriction site was altered without changing the encoded amino acid(TCT=Serin (TC(T,A,G,C)).

Primers for Pfu-mutagenese:
Forward primer (5’->3’) (24 bp)
GCAAACTCTTCAAGAGCTAGAGAG

Reverse primer (5’->3’) (24 bp)
CTCTCTAGCTCTTGAAGAGTTTGC

Phytochrome Interacting Factor-6 (PIF6) - BBa_K365001


Background:

For the design of the first engineered system that achieved to enable the spatiotemporal control of PhyB-PIF interactions in in-vivo experiments, [42]. screened multiple potential phytochrome–PIF pairs by a fluorescence translocation assay in NIH3T3 cells. They measured the red-light-induced translocation of yellow fluorescent protein (YFP) fused to PIF domains to co-expressed phytochrome domains fused through a flexible linker to mCherry and localized to the plasma membrane by a carboxyterminalpolybasic, prenylation sequence from Kras. Of all previously reported PIF domains, only the N terminus of PIF6 is strong enough to cause significant translocation of YFP to the membrane.

Fig.1: Implied system of Lim and Voigt (2009) to screen potential phytochrome-PIF
pairs in a fluorescence translocation assay.


Conception:

We chose used the same sequence of the last 100-residue N-terminal activated phytochrome binding (APB) domain of PIF6, which was already successfully used by [42]..

There is no PDB structure for the PIF6
The stucture below is modeled by homology using the server I-TASSER


The plasmid containing the PIF6-sequence was provided by the laboratory of Wilfried Weber from the University of Freiburg. For the synthesis of the Pif6 BioBrick primers containing the sites of the Fusion Protein BioBrick Assembly Standard were used.

Forward primer (5’->3’): 54 bp
GGATCCgaattcgcggccgcttctagatggccggcATGATGTTCTTACCAACCG

Reverse primer (5’->3’): 58 bp
CAGCTGctgcagcggccgctactagtattaaccggtGTCAACATGTTTATTGCTTTCC

∆N-ClpX (aa 61-425) - BBa_K365004


Background

ClpXP is a part of an E.coli protease which consists of three parts, the hexametric ClpX and two heptametrical ClpP subunits. ClpX consists of six identical subunits, each 1092bp long. ClpX recognizes and unfolds protein containing certain tags like LAA and leading them into the catalytic center of this protein complex, the two ClpP units. ClpX has two internal restriction sides for EcoRI and two restriction sides for AgeI.

Conception

ClpX has two internal restriction sides for EcoRI and two restriction sides for AgeI. The purpose of this first experimental part was to extract the ClpX gene out of the E.coli genome, to alter the internal EcoRI and AgeI sides in the ClpX gene and to fuse iGEM fusion pre- and suffixes to the ClpX sequence in order to get an iGEM Biobrick with standard prefix and suffix standard without internal EcoRI, Not, XbaI, AgeI, SpeI and PstI sides.

The sequence was obtained from the following database for DH5α E.coli cells:

1- http://www.ncbi.nlm.nih.gov/gene/945083

2- http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=456650&to=457924&report=gbwithparts




Problem: 2 AgeI and 2 EcoRI sides

Primers for cloning ClpX out of the E.Coli genome.
These primes were used to amplificate ClpX from the E.coli genome.

Forward primer (5’->3’) : 31bp
CGCAGTGCGCTACCGACGCCGCATGAAATTC

Reverse primer (5’->3’) : 32bp
TTCACCAGATGCCTGTTGCGCTTCCGGCTTGC

Primers for Pfu-mutagenese

1. ACC=Thr AC(A,T,G,C), GGT=Gly GG(A,T,G,C)       AgeI site


Forward primer (5’->3’) (31 bp)
CTGATCGGTCCGACTGGTTCCGGTAAAACGC

Reverse primer (5’->3’) (31 bp)
GCGTTTTACCGGAACCAGTCGGACCGATCAG

2. GAA=Glu GA(A,G), TTC=Phe TT(C,T)       EcoRI site


Forward primer (5’->3’) (28 bp)
CATCCGCAGCAGGAGTTCTTGCAGGTTG

Reverse primer (5’->3’) (28 bp)
CAACCTGCAAGAACTCCTGCTGCGGATG

3. GAA=Glu GA(A,G), TTC=Phe TT(C,T)       EcoRI site


Forward primer (5’->3’) (25 bp)
CGTGGATCTGGAGTTCCGTGACGAG

Reverse primer (5’->3’) (25 bp)
CTCGTCACGGAACTCCAGATCCACG

4. ACC=Thr AC(A,T,G,C), GGT=Gly GG(A,T,G,C)       AgeI site


Forward primer (5’->3’) (24 bp)
GGCGCGTAAAACTGGTGCCCGTGG

Reverse primer (5’->3’) (24 bp)
CCACGGGCACCAGTTTTACGCGCC

Primers for amplification of ClpX with fusion pre- and suffix
After mutagenesis of internal restriction sides, the fusion pre- and suffixes were added to the ClpX gene.

Forward primer (5’->3’):
GGATCCgaattcgcggccgcttctagatggccggcCGCAGTGCGCTACCGACGCCGC

Reverse primer (5’->3’):
CAGCTGctgcagcggccgctactagtattaaccggtTTCACCAGATGCCTGTTGCGC


Linker (aa 20) - BBa_K365005


Background:

The linker biobrick is used to join the three ClpX subunits covalently in order to build a ClpX trimer and to link the degradation tags and PIF3/6 with the protein destined for degradation.

Conception:

We chose to use the same linker, which was already successfully used by [2] to construct the ClpX trimer. It is a twenty amino acid linker (ASGAGGSEGGGSEGGTSGAT). The codon usage of E. coli (http://www.geneinfinity.org/sp_codonusage.html) has been used to decide the DNA sequence, in addition RFC 25 fusion prefix and suffix have been added to the sequence.



The linker has been order as six separate, EcoRI+AgeI precut primers, which were hybridized in order to obtain the complete linker sequence.

Forward strand (5’->3’) : (89 bp)
aattcgcggccgcttctagatggc|cggcGCGAGCGGCGCGGGCGGCAGCGAAGGCGGCGGCAG|CGAAGGCGGCACCAGCGGCGCGACCa

Reverse strand (5’-3’) : (89 bp)
ccggtGGTCGCGCCG|CTGGTGCCGCCTTCGCTGCCGCCGCCTTCGCTGCC|GCCCGCGCCGCTCGCgccggccatctagaagcggccgcg

Ordered primer:

Forward:         5’aattcgcggccgcttctagatggc 3’

Forward:         5’cggcGCGAGCGGCGCGGGCGGCAGCGAAGGCGGCGGCAG 3’

Forward:         5’CGAAGGCGGCACCAGCGGCGCGACCa 3’

Reverse:          5’ccggtGGTCGCGCCG 3’

Reverse:          5’CTGGTGCCGCCTTCGCTGCCGCCGCCTTCGCTGCC 3’

Reverse:          5’GCCCGCGCCGCTCGCgccggccatctagaagcggccgcg3’


LAA tag - BBa_K365006


Background:

LAA tag is a C-terminal region of the natural ssrA-recognition sequence of E. Coli that interacts with the ClpXP protease. A protein fused with this tag will be preferentially degraded by the ClpX protease without need of an adaptor protein [6]. This tag serves as positive control for the functionality of the composed ClpXP and the PhyB-ClpXP fusion protein.

Conception:

We chose to use the same tag, which was already successfully used by used by [6]. It is a eleven amino acid tag (AANDENYALAA). At the end of the coding sequence a double stop codon was added (ACTAGT). The codon usage of E. coli (http://www.geneinfinity.org/sp_codonusage.html) has been used to decide the DNA sequence and NgoMIV and Pst1 restriction sites have been added to the sequence.

The LAA-tag has been order as two separate, NgoMIV + Pst1 precut primers, which were hybridized in order to obtain the degradation sequence.

LAA Tag


Ordered primer

Forward:         5’CCGGCGCGCTGGCGGCGTAATAATACTAGTAGCGGCCGC3’

Reverse:          5’ TGCAGCGGCCGCTACTAGTATTATTACGCCGCCAGCGCG 3’



For the synthesis of the PstI-site a mistake occurred in the command of the primers, as we did not consider that PstI cuts in the (3’ -> 5’) sens, contrary to the other restriction enzymes of the BioBrick standard. A supplementary step of ligation digestion in the experimental procedure can fix this mistake.


DAS tag - BBa_K365007

Background:

The DAS tag presents a C-terminal recognition sequence that has been artificially altered so that it has weakened interactions with ClpXP and depends on an adaptor [6]. In E. coli, the adaptor SspB tethers specifically tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. In our system, the role of the adaptor-protein SspB has been assumed by Pif3/6. So only light-induced activation can lead to binding and efficient degradation of DAS bearing constructs.

Conception:

We decided to use the same tag, which was already successfully used by [6]. It is a eleven amino acid tag (AANDENYADAS). At the end of the coding sequence a double stop codon was added (ACTAGT). The codon usage of E. coli (http://www.geneinfinity.org/sp_codonusage.html) has been used to decide the DNA sequence, in addition NgoMIV and PST1 restriction sites have been added to the sequence.

The DAS-tag has been order as two separate, NgoMIV + Pst1 precut primers, which were hybridized in order to obtain the degradation sequence.



Ordered primer

Forward:         5’ CCGGCGCGGATGCGAGCTAATAATACTAGTAGCGGCCGC3’

Reverse:          5’ TGCAGCGGCCGCTACTAGTATTATTAGCTCGCATCCGCG 3’



For the synthesis of the PstI-site a mistake occurred in the command of the primers, as we did not consider that PstI cuts in the (3’ -> 5’) sens, contrary to the other restriction enzymes of the BioBrick standard. A supplementary step of ligation digestion in the experimental procedure can fix this mistake.


Lambda tag - BBa_K365008


Background:

The λO- tag is the N-terminal equalent to the DAS tag. Degradation of proteins bearing the N-terminal λO- tag normally requires the N-domain of ClpX, which is missing in the PhyB-linker-[ClpX]3 variant.

[2] used this tag to test an artificial tethering system and demonstrated that it can serve as degradation signal for substrates that are tethered to ClpX.

Conception:

We chose to use the same sequence, which was already successfully used by used by [2]: NH2-TNTAKILNFGR. The codon usage of E. coli (http://www.geneinfinity.org/sp_codonusage.html) has been used to decide the DNA sequence, in addition NgoMIV and AgeI restriction sites have been added to the sequence.

The tag has been order as two separate, NgoMIV + Pst1 precut primers, which were hybridized in order to obtain the whole degradation tag.



Ordered primer

Forward:         5’AATTCGCGGCCGCTTCTAGATGACCAACACCGCGAAAATTCTGAACTTTGGCCGCA 3’

Reverse:          5’CCGGTGCGGCCAAAGTTCAGAATTTTCGCGGTGTTGGTCATCTAGAAGCGGCCGCG 3’




GFP (super fold) - BBa_K365009


Background

The GFP super fold protein originated from the BBa_I746915. This old biobrick contains the normal standard prefix and suffix. When this GFP is now fused to another biobrick a stop codon is created which would terminate the transcription of the fused biobrick. To avoid this inconvenience, we improved this biobrick to the fusion standard BBF RFC 25. This new biobrick can now be added to any biobrick with compatible restriction sides without creating a stop codon.


Conception

Standard PCR techniques were used. The Primer for amplification contained the fusion pre- and suffix and compatible base pair for the GFP gene.

PhyB642-(linker-∆NClpX)3 - BBa_K365010


Background:

This final construct will be the inducible part of the protease system. This permits the linking of the phytochrome B to the Clpx trimer which forms the light-dependent activation part of our degradation system.



Conception:

The standards cloning methods were used. This includes digestion of the vector containing linker- ClpX trimer and the PhyB part then the ligation of them and to finish the transformation of competente cells with this plasmid.


PhyB908-(linker-∆NClpX)3 - BBa_K365011


Background:

This final construct will be the inducible part of the protease system. This permits the linking of the phytochrome B to the Clpx trimer which forms the light-dependent activation part of our degradation system.



Conception:

The standards cloning methods were used. This includes digestion of the vector containing linker- ClpX trimer and the PhyB part then the ligation of them and to finish the transformation of competente cells with this plasmid.


Full-length ClpX - BBa_K365012


Background

ClpXP is a part of an E.coli protease which consists of three parts, the hexametric ClpX and two heptametrical ClpP subunits. ClpX consists of six identical subunits, each 1092bp long. ClpX recognizes and unfolds protein containing certain tags like LAA and leading them into the catalytic center of this protein complex, the two ClpP units. ClpX has two internal restriction sides for EcoRI and two restriction sides for AgeI.

Conception

ClpX has two internal restriction sides for EcoRI and two restriction sides for AgeI. The purpose of this first experimental part was to extract the ClpX gene out of the E.coli genome, to alter the internal EcoRI and AgeI sides in the ClpX gene and to fuse iGEM fusion pre- and suffixes to the ClpX sequence in order to get an iGEM Biobrick with standard prefix and suffix standard without internal EcoRI, Not, XbaI, AgeI, SpeI and PstI sides.

The sequence was obtained from the following database for DH5α E.coli cells:

1- http://www.ncbi.nlm.nih.gov/gene/945083

2- http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=456650&to=457924&report=gbwithparts




Problem: 2 AgeI and 2 EcoRI sides

Primers for cloning ClpX out of the E.Coli genome.
These primes were used to amplificate ClpX from the E.coli genome.

Forward primer (5’->3’) : 31bp
CGCAGTGCGCTACCGACGCCGCATGAAATTC

Reverse primer (5’->3’) : 32bp
TTCACCAGATGCCTGTTGCGCTTCCGGCTTGC

Primers for Pfu-mutagenese

1. ACC=Thr AC(A,T,G,C), GGT=Gly GG(A,T,G,C)       AgeI site


Forward primer (5’->3’) (31 bp)
CTGATCGGTCCGACTGGTTCCGGTAAAACGC

Reverse primer (5’->3’) (31 bp)
GCGTTTTACCGGAACCAGTCGGACCGATCAG

2. GAA=Glu GA(A,G), TTC=Phe TT(C,T)       EcoRI site


Forward primer (5’->3’) (28 bp)
CATCCGCAGCAGGAGTTCTTGCAGGTTG

Reverse primer (5’->3’) (28 bp)
CAACCTGCAAGAACTCCTGCTGCGGATG

3. GAA=Glu GA(A,G), TTC=Phe TT(C,T)       EcoRI site


Forward primer (5’->3’) (25 bp)
CGTGGATCTGGAGTTCCGTGACGAG

Reverse primer (5’->3’) (25 bp)
CTCGTCACGGAACTCCAGATCCACG

4. ACC=Thr AC(A,T,G,C), GGT=Gly GG(A,T,G,C)       AgeI site


Forward primer (5’->3’) (24 bp)
GGCGCGTAAAACTGGTGCCCGTGG

Reverse primer (5’->3’) (24 bp)
CCACGGGCACCAGTTTTACGCGCC



∆NClpX-linker-∆NClpX-linker-∆NClpX - BBa_K365013


Background:

Phytochrome B needs to be fixed to the Clpx hexametric part of the protease. It is possible to link one phytochrome B per ClpX monomer but this could lead to steric problems. So the decision was made to follow the idea of the publication of Tanja Baker 2009. This permits to have only one phytochrome B for three ClpX units. Moreover the publication proved that the speed of the assembly of two ClpX trimers is quite the same than with ClpX monomers. So the Clpx hexameric part is composed of two ClpX trimers each one couple with a phytochrome B.



Conception: ClpX primer was constructed due to cloning methods. The new approach in this method is that the primer was built from a PCR product. The construction of the ClpX dimer was conducted with standard methods. Then in a PCR reaction the ClpX primers located inside of the dimer were used to construct this new primer. The product of this PCR was used as primer for constructing the ClpX trimer.



(linker-∆NClpX)3 - BBa_K365014


Background:

This construct was the final step for the ClpX hexamer creation. The two trimers will assemble to the full ClpX unit. Then the phytochrome B was cloned to this trimer. It will also be possible to add any other activation system like hormone receptors to this biobrick instead of phytochrome B, giving this part a huge potential of further applications.



Conception:

The standards cloning methods were used. This includes digestion of the vector containing ClpX trimer and the linker part then the ligation of them and to finish the transformation of competente cells with this plasmid.