Difference between revisions of "Team:Ecuador/Parts"

 
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<div class="ec--first--section">
 
<div class="ec--first--section">
 
<div class="ec--main--title">
 
<div class="ec--main--title">
−
<a name="INTER"  style="text-decoration: none;">
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<a name="MOD"  style="text-decoration: none;"><h1>PARTS</h1></a>
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<div class="ec--h1">
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−
INTERLAB
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</div>
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</a>
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</div>
 
</div>
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<center>
 
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  <p><img width="385" height="244" src="https://static.igem.org/mediawiki/2018/d/d6/T--ECUADOR--Modeling.jpeg"></p></center>
 
 
<div class="ec--modeling--items">
 
<div class="ec--modeling--items">
−
<ul>
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<h3>Index</h3>
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<li><a href="#WWD">
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<ul class="circle">
−
WHAT DID WE DO?
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<a href="#INT" style="text-decoration: none;color: #000000"><li>Introduction</li></a>
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</a>
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<a href="#BAC" style="text-decoration: none;color: #000000"><li>Bone and cartilage regeneration</li></a>
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</li>
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<a href="#ELA" style="text-decoration: none;color: #000000"><li>Elasticity</li></a>
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<li><a href="#HWD">
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<a href="#CELL" style="text-decoration: none;color: #000000"><li>Cellulose production </li></a>
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HOW DID WE DO IT?
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</a>
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</li>
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<li><a href="#BIO">
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BioBricks
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</a>
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</li>
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<li><a href="#RES">
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RESULTS
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</a>
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</li>
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<li><a href="#EXP">
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EXPERIENCE
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</a>
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</li>
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−
<li><a href="#ACK">
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ACKNOWLEDGEMENT
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</a>
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</li>
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</ul>
 
</ul>
 
</div>
 
</div>
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<div class="ec--nau--content">
 
<div class="ec--nau--content">
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  <a name="WWD">
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<a name="INT"></a>
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<div class="ec--h2">
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<div class="ec--h2">
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What did we do?
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INTRODUCTION
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</div>
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</div>
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</a>
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<div class="ec--p">
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                <div class="ec--p">
−
We tried to reduce the variability in the measurement of GFP expression for this reason we used a direct method to determine the mean expression level of GFP per cel
+
The aim of our project is to obtain a novel bone and cartilage regeneration treatment. The path we take to carry out this was transforming E. coli to produce a fusion protein, conformed by a cellulose binding domain and a protein that induce chondrogenesis and osteogenesis, also a fusion protein conformed by CBD and an elastin like protein was design in order to obtain an elastic biomaterial. We also tried to produce cellulose in E. coli to have a better control in its production with a LacI repressed promoter. The parts are briefly described below (see the part page if more information is needed).
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</div>
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  <a name="HWD">
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<div class="ec--h2">
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How did we do it?
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</div>
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</a>
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<div class="ec--p">
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−
In order to avoid distortions in the results due to differences in the experimentation processes, we follow the procedures described in the iGEM protocol (<a href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf" style="text-decoration: none">Plate reader protocol</a>, <a href="http://parts.igem.org/Help:Protocols/Competent_Cells" style="text-decoration: none">Competent cells</a>, <a href="http://parts.igem.org/Help:Protocols/Transformation" style="text-decoration: none">Transformation</a>)l
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</div>
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−
+
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  <div class="ec--h3">
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−
Calibration
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</div>
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−
<div class="ec--p">Before starting the experiments, we performed the OD600 calibration measurements following the LUDOX protocol, the particle Estandar Curve with the Microsphere Protocol, and the fluorescence standard curve with the Fluorescein Protocol. This allowed us to familiarize ourselves with the plate reader required for subsequent measurements. The plate reader that we used had the following characteristics:<br />
+
−
  Label: BioTek / Model: Cytation 5 / Emission: 528 / Excitation: 485 / Optic position: Top / Temperature: 25 ºC / Shake: 425 cpm for 30 sec before every measurement.
+
 
</div>
 
</div>
−
+
<a name="BAC"></a>
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<div class="ec--h3">Cell measurement</div>
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<div class="ec--h2">
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<div class="ec--p">
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Fusion protein for bone and cartilage regeneration
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We started by transforming E. coli DH5 alpha with the plasmid described in the protocol, and after incubation and growth in LB-medium, we diluted the culture to Abs600 0.2 and proceeded  with the samples.
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−
</div>
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−
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−
<div class="ec--h3">Colony units per 0.1 OD600 E. coli cultures</div>
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−
<div class="ec--p">
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−
In order to determine the cell concentration of the culture we followed the CFU protocol that basically consisted in diluting a culture to certain absorbance, measure the OD600 that has to be in a value of 0.1, and make serial dilutions to later count the colonies and calculate the CFU/mL.
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−
</div>
+
−
+
−
  <a name="BIO"><div class="ec--h2">BioBricks</div></a>
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−
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<div class="ec--biob--cont">
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<div id="bigBox">
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<div id="leftBox">
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    <table border="0" cellspacing="8px" cellpadding="0">
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          <tr>
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−
            <td width="148" valign="top"><center ><strong>Device</strong><strong> </strong></center></td>
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−
            <td width="132" valign="top"><center><strong>Part Number</strong><strong> </strong></center></td>
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−
              </tr>
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          <tr>
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−
            <td width="148" valign="top"><p id="label-neg-c" class="ec--inter-item">Negative control</p></td>
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−
            <td width="132" valign="top"><p>BBa_R0040 </p></td>
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−
              </tr>
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−
          <tr>
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            <td width="148" valign="top"><p id="type"class="ec--inter-item">Positive control </p></td>
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            <td width="132" valign="top"><p>BBa_I20270 </p></td>
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−
              </tr>
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−
          <tr>
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            <td width="148" valign="top"><p id="label-td1" class="ec--inter-item">Test Device 1 </p></td>
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            <td width="132" valign="top"><p>BBa_J364000 </p></td>
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              </tr>
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−
          <tr>
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−
            <td width="148" valign="top"><p id="label-td2" class="ec--inter-item">Test Device 2 </p></td>
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−
            <td width="132" valign="top"><p>BBa_J364001 </p></td>
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−
              </tr>
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−
          <tr>
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−
            <td width="148" valign="top"><p id="label-td3" class="ec--inter-item">Test Device 3 </p></td>
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−
            <td width="132" valign="top"><p>BBa_J364002 </p></td>
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−
              </tr>
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−
          <tr>
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−
            <td width="148" valign="top"><p id="label-td4" class="ec--inter-item">Test Device 4 </p></td>
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−
            <td width="132" valign="top"><p>BBa_J364007 </p></td>
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−
              </tr>
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−
          <tr>
+
−
            <td width="148" valign="top"><p id="label-td5" class="ec--inter-item">Test Device 5 </p></td>
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−
            <td width="132" valign="top"><p>BBa_J364008 </p></td>
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−
              </tr>
+
−
          <tr>
+
−
            <td width="148" valign="top"><p id="label-td6" class="ec--inter-item">Test Device 6 </p></td>
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−
            <td width="132" valign="top"><p>BBa_J364009 </p></td>
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−
              </tr>
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−
            </table>
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−
  </div>
+
−
<div id="rightBox">
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−
<div class="ec--biobr--location--container">
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−
<table width="338" border="0" cellpadding="0" cellspacing="8px">
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−
  <tr>
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−
<td width="330" valign="top"><p align="center"><strong>Structure<br>
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−
(Point with the cursor on the device)
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−
</strong></p></td>
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−
  </tr>
+
−
</table>
+
−
<div class="ec--biobr--separator"></div>
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−
<div class="loc-neg-c">
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−
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−
<p><img width="359" height="75" src="https://static.igem.org/mediawiki/2018/c/c9/T--ECUADOR--L001.png"> </p>
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−
<br>
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−
<p>length: 54 bp </p>
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−
  </div>
+
−
<div class="textdis" id="pos_c">
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−
+
−
<p><img width="195" height="70" src="https://static.igem.org/mediawiki/2018/b/b5/T--ECUADOR--L002.png"></p>
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<br>
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<p>length: 919 bp</p>
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</div>
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−
<div class="loc-td1">
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−
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<p><img width="195" height="51" src="https://static.igem.org/mediawiki/2018/8/86/T--ECUADOR--L003.png" > </p>
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<br>
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−
<p>length: 918 bp </p>
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−
</div>
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−
<div class="loc-td2">
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−
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<p><img width="189" height="54" src="https://static.igem.org/mediawiki/2018/c/c8/T--ECUADOR--L004.png" ></p>
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<br>
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−
<p>length: 918 bp</p>
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</div>
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−
<div class="loc-td3">
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−
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<p><img width="197" height="63" src="https://static.igem.org/mediawiki/2018/4/43/T--ECUADOR--L005.png" > </p>
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<br>
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<p>length: 918 bp </p>
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−
</div>
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−
<div class="loc-td4">
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−
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<p><img width="196" height="52" src="https://static.igem.org/mediawiki/2018/5/53/T--ECUADOR--L006.png"></p>
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<br>
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−
<p>length: 918 bp</p>
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−
</div>
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−
<div class="loc-td5">
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−
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<p><img width="188" height="64" src="https://static.igem.org/mediawiki/2018/9/97/T--ECUADOR--L007.png"  > </p>
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<br>
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<p>length: 918 bp </p>
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</div>
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−
<div class="loc-td6">
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−
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<p><img width="194" height="55" src="https://static.igem.org/mediawiki/2018/0/02/T--ECUADOR--L008.png"></p>
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<br>
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<p>length: 918 bp</p>
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</div>
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−
    </div>
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</div>
 
</div>
−
  </div>
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 +
<div class="ec--h3">Coding parts</div>
 +
 +
<div class="ec--BMP--container">
 +
<a href="http://parts.igem.org/Part:BBa_K2804000" style="color:black;"><div class="ec--h4" style="color:black;">BBa_K2804000   
 +
 +
      </div></a>
 +
<strong>CBD cipA with a C-terminal linker</strong>
 +
<div class="ec--BMP--text">
 +
    <div class="ec--p">This part was design to try to improve the imperial college (2014) CBD cipA part  BBa_K1321014, because the CBD N-terminal is where the protein binds to the cellulose and the linker could interfer with this interaction.</div>
 +
  </div>
 
  </div>
 
  </div>
−
+
−
<a name="RES"><div class="ec--h2">Results</div></a>
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<div class="ec--BMP--container">
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  <div class="ec--h3">Particle standard curves</div>
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<a href="http://parts.igem.org/Part:BBa_K2804006" style="color:black;"><div class="ec--h4">
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<div class="ec--img--wwd--cont">
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BBa_K2804006 
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<img src="https://static.igem.org/mediawiki/2018/3/30/T--ECUADOR--A-PSC.1.png" width="537" height="307" alt=""/>
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</div>
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      </div>
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<div class="ec--img--wwd--cont">
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</a>
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<img src="https://static.igem.org/mediawiki/2018/1/1d/T--ECUADOR--A-PSC.2.png" width="534" height="303" alt=""/>
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<strong>Superfolder GFP with six-histidine tail</strong>
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</div>
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<div class="ec--BMP--text">
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    <div class="ec--p">The adition of an histidine tail to part BBa_K1365020 was done because it allows the characterization of fusion proteins binding sites through atomic force microscopy, thanks to its affinity to the equipment nickel tip.</div>
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<div class="ec--h3">Fluorescein standard curves</div>
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  </div>
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<div class="ec--img--wwd--cont">
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    <p><img src="https://static.igem.org/mediawiki/2018/7/7c/T--ECUADOR--A-FSC.1.png" width="534" height="303" alt=""/><br></p>
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  </div>
 
  </div>
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              <div class="ec--img--wwd--cont">
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  <img src="https://static.igem.org/mediawiki/2018/c/cf/T--ECUADOR--A-FSC.2.png" width="534" height="303" alt=""/><br>
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<div class="ec--BMP--container">
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  </div>
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<a href="http://parts.igem.org/Part:BBa_K2804002" style="color:black;"><div class="ec--h4">BBa_K2804002         
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                <div class="ec--h3">Converting between absorbance of cells to absorbance of a known concentration of beads</div>
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      </div></a>
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<div class="ec--img--wwd--cont">
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<strong>Bone morphogenetic protein 2 (BMP2)</strong>
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              <p><img src="https://static.igem.org/mediawiki/2018/6/64/T--ECUADOR--A-NA600.png" width="534" height="303" alt=""/></p>
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<div class="ec--BMP--text">
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</div>
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    <div class="ec--p">
−
  <div class="ec--h3">Counting colony-forming units (CFUs) from the sample</div>
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    Bone morphogenetic protein 2 (BMP2), is a member of the transforming growth factor-β (TGF-β) super-family. It is involved in chondrogenesis and osteogenesis in vitro via the expression induction of Sox9, Runx2 and its downstream markers.<sup>1</sup>
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<div class="ec--img--wwd--cont">
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              <p><img src="https://static.igem.org/mediawiki/2018/2/2e/T--ECUADOR--A-RBT.png" width="534" height="303" alt=""/></p>
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    </div>
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</div>
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  </div>
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  <a name="EXP"><div class="ec--h2">EXPERIENCE</div></a>
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<div class="ec--h3">Composite parts</div>
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</a>
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              <div class="ec--img--wwd--cont">
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<div class="ec--BMP--container">
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                <center><img src="https://svgsilh.com/svg/2324013-2196f3.svg" width="309" height="175" alt=""/></center>
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<a href="http://parts.igem.org/Part:BBa_K2804001" style="color:black;"><div class="ec--h4">BBa_K2804001 
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              </div>
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      </div></a>
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              <div class="ec--p">The interlab protocols were easy to understand and simple. We recommend the following:</div>
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<strong>CBD cipA fused to BMP2 under the control of LacI promoter</strong>
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              <ul>
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<div class="ec--BMP--text">
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                <li>
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    <div class="ec--p">Therapeutic protein coupled to the CBD  whose expression is regulated by the presence of IPTG in the medium.</div>
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                  <div class="ec--p">Before each measurement, shake the plates in the equipment since the particles tend to precipitate in the well.</div>
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  </div>
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                </li>
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  </div>
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                <li dir="ltr">
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<div class="ec--BMP--container">
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                  <div class="ec--p">If the iGEM equipment does not have the plate reader in the same place as your laboratory, they must take care of their samples in continuous refrigeration during transport and carry out the activities with time.</div>
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<a href="http://parts.igem.org/Part:BBa_K2804004" style="color:black;"><div class="ec--h4">BBa_K2804004
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                </li>
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      </div></a>
−
              </ul>
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CBD cipA fused to sfGFP
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              <div class="ec--p">We learned many things in the interlab challenge: the protocols, the management of the plate reader and how it can be used in later processes of our project. It also helped us strengthen cooperation with other universities.</div>
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<div class="ec--BMP--text">
−
+
  <div class="ec--p"></div>
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              <a name="ACK" style="text-decoration: none;">
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</div>
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              <div class="ec--h2">ACKNOWLEDGEMENT</div>
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</div>
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              </a>
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<div class="ec--p">
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<div class="ec--BMP--container">
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As a team, we would like to thank the  Biomedicine team of Universidad Tecnica Equinoccial and especially Linda  Guaman, member of their research team, for allowing us to use their measurement  equipment, which were essential for the completion of the Interlab.</div>
+
<a href="http://parts.igem.org/Part:BBa_K2804005" style="color:black;"><div class="ec--h4">BBa_K2804005
−
+
      </div></a>
 +
CBD cipA fused to sfGFP under the control of LacI promoter
 +
<div class="ec--BMP--text">
 +
  <div class="ec--p"></div>
 +
</div>
 +
</div>
 +
<div class="ec--BMP--container">
 +
<a href="http://parts.igem.org/Part:BBa_K2804014" style="color:black;"><div class="ec--h4">BBa_K2804014
 +
      </div></a>
 +
CBD cipA fused to sfGFP
 +
<div class="ec--BMP--text">
 +
  <div class="ec--p"></div>
 +
</div>
 +
</div>
 +
  <div class="ec--BMP--container">
 +
<a href="http://parts.igem.org/Part:BBa_K2804015" style="color:black;"><div class="ec--h4">BBa_K2804015
 +
      </div></a>
 +
CBD cipA fused to BMP2
 +
<div class="ec--BMP--text">
 +
  <div class="ec--p"></div>
 +
</div>
 +
</div>
 +
<table border="0" cellspacing="0" cellpadding="0" width="703">
 +
  <tr>
 +
    <td width="175" nowrap valign="bottom"><p><strong>Name</strong></p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>Description</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p><strong>Type</strong></p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804000</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>CBD cipA with a C-terminal linker</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Coding</p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804001</p></td>
 +
    <td width="448" nowrap valign="bottom"><p>CBD cipA fused to BMP2 under the control of LacI    promoter</p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Composite</p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804002</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>Bone morphogenetic protein 2 (BMP2)</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Coding</p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804004</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>CBD cipA fused to sfGFP</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Composite</p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804005</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>CBD cipA fused to sfGFP under the control    of LacI promoter</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Composite</p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804006</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>Superfolder GFP with six-histidine tail</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Coding</p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804014</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>CBD cipA fused to sfGFP</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Composite</p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804015</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>CBD cipA fused to BMP2</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Composite</p></td>
 +
      </tr>
 +
  </table>
 +
<p>&nbsp;</p>
 +
 +
<a name="ELA"></a>
 +
<div class="ec--h2">
 +
Fusion protein to provide elasticity to the biomaterial
 +
</div>
 +
 +
<div class="ec--h3">Coding parts</div>
 +
 +
<div class="ec--BMP--container">
 +
  <a href="http://parts.igem.org/Part:BBa_K2804011" style="color:black;"><div class="ec--h4">BBa_K2804011   
 +
      </div></a>
 +
ELP V-150
 +
<div class="ec--BMP--text">
 +
  <div class="ec--p"></div>
 +
</div>
 +
</div>
 +
<div class="ec--BMP--container">
 +
  <a href="http://parts.igem.org/Part:BBa_K2804012" style="color:black;"><div class="ec--h4">BBa_K2804012 
 +
      </div></a>
 +
ELP V-150
 +
<div class="ec--BMP--text">
 +
  <div class="ec--p"></div>
 +
</div>
 +
</div>
 +
 +
<div class="ec--h3">Composite part</div>
 +
 +
  <div class="ec--BMP--container">
 +
<a href="http://parts.igem.org/Part:BBa_K2804013" style="color:black;"><div class="ec--h4">BBa_K2804013   
 +
      </div></a>
 +
CBD cipA fused to ELP_C5 under the control of LacI promoter
 +
<div class="ec--BMP--text">
 +
  <div class="ec--p"></div>
 +
</div>
 +
</div>
 +
<table border="0" cellspacing="0" cellpadding="0" width="703">
 +
  <tr>
 +
    <td width="175" nowrap valign="bottom"><p><strong>Name</strong></p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>Description</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p><strong>Type</strong></p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804011</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>ELP V-150</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Coding</p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804012</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>ELP_C5</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Coding</p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804013</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>CBD cipA fused to ELP_C5 under the control    of LacI promoter</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Composite</p></td>
 +
      </tr>
 +
  </table>
 +
<p>&nbsp;</p>
 +
 +
<a name="CELL"></a>
 +
<div class="ec--h2">
 +
Cellulose production module
 +
</div>
 +
 +
<div class="ec--h3">Coding parts</div>
 +
 +
<div class="ec--BMP--container">
 +
  <a href="http://parts.igem.org/Part:BBa_K2804008" style="color:black;">
 +
  <div class="ec--h4">
 +
  BBa_K2804008 
 +
      </div>
 +
  </a>
 +
CmcAx: Endo-beta-1,4-glucanase ezyme
 +
<div class="ec--BMP--text">
 +
  <div class="ec--p"></div>
 +
</div>
 +
</div>
 +
<div class="ec--BMP--container">
 +
  <a href="http://parts.igem.org/Part:BBa_K2804009" style="color:black;"><div class="ec--h4">BBa_K2804009 
 +
      </div></a>
 +
CcpAx: Cellulose-complementing protein
 +
<div class="ec--BMP--text">
 +
  <div class="ec--p"></div>
 +
</div>
 +
</div>
 +
  </div>
 +
 +
<div class="ec--h3">Composite part</div>
 +
 +
<div class="ec--BMP--container">
 +
  <a href="http://parts.igem.org/Part:BBa_K2804010" style="color:black;"><div class="ec--h4">BBa_K2804010 
 +
      </div></a>
 +
Operon member of bacterial cellulose synthase complex: Cmcax and CcpAx
 +
<div class="ec--BMP--text">
 +
  <div class="ec--p"></div>
 +
</div>
 +
</div>
 +
<table border="1" cellspacing="0" cellpadding="0" width="703">
 +
  <tr>
 +
    <td width="175" nowrap valign="bottom"><p><strong>Name</strong></p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>Description</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p><strong>Type</strong></p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804008</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>CmcAx: Endo-beta-1,4-glucanase ezyme</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Coding</p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804009</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>CcpAx: Cellulose-complementing protein</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Coding</p></td>
 +
      </tr>
 +
  <tr>
 +
    <td width="175"><p>BBa_K2804010</p></td>
 +
    <td width="448" nowrap valign="bottom"><p><strong>Operon member of bacterial cellulose    synthase complex: Cmcax and CcpAx</strong></p></td>
 +
    <td width="80" nowrap valign="bottom"><p>Composite</p></td>
 +
      </tr>
 +
</table>
 
</div>
 
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</body>
 
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+
<div class="ec--h4"><a name="RE"></a>REFERENCES</div>
 +
<div class="ec--p">
 +
<div class="ec--p">Zhou,  N., Li, Q., Lin, X. et al. Cell Tissue Res (2016) 366: 101. <a href="https://doi.org/10.1007/s00441-016-2403-0">https://doi.org/10.1007/s00441-016-2403-0</a></div>
 +
  </div>
 +
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Latest revision as of 02:32, 19 November 2018

iGEM ECUADOR

PARTS

Index

INTRODUCTION
The aim of our project is to obtain a novel bone and cartilage regeneration treatment. The path we take to carry out this was transforming E. coli to produce a fusion protein, conformed by a cellulose binding domain and a protein that induce chondrogenesis and osteogenesis, also a fusion protein conformed by CBD and an elastin like protein was design in order to obtain an elastic biomaterial. We also tried to produce cellulose in E. coli to have a better control in its production with a LacI repressed promoter. The parts are briefly described below (see the part page if more information is needed).
Fusion protein for bone and cartilage regeneration
Coding parts
BBa_K2804000
CBD cipA with a C-terminal linker
This part was design to try to improve the imperial college (2014) CBD cipA part BBa_K1321014, because the CBD N-terminal is where the protein binds to the cellulose and the linker could interfer with this interaction.
BBa_K2804006
Superfolder GFP with six-histidine tail
The adition of an histidine tail to part BBa_K1365020 was done because it allows the characterization of fusion proteins binding sites through atomic force microscopy, thanks to its affinity to the equipment nickel tip.
BBa_K2804002
Bone morphogenetic protein 2 (BMP2)
Bone morphogenetic protein 2 (BMP2), is a member of the transforming growth factor-β (TGF-β) super-family. It is involved in chondrogenesis and osteogenesis in vitro via the expression induction of Sox9, Runx2 and its downstream markers.1
Composite parts
BBa_K2804001
CBD cipA fused to BMP2 under the control of LacI promoter
Therapeutic protein coupled to the CBD whose expression is regulated by the presence of IPTG in the medium.
BBa_K2804004
CBD cipA fused to sfGFP
BBa_K2804005
CBD cipA fused to sfGFP under the control of LacI promoter
BBa_K2804014
CBD cipA fused to sfGFP
BBa_K2804015
CBD cipA fused to BMP2

Name

Description

Type

BBa_K2804000

CBD cipA with a C-terminal linker

Coding

BBa_K2804001

CBD cipA fused to BMP2 under the control of LacI promoter

Composite

BBa_K2804002

Bone morphogenetic protein 2 (BMP2)

Coding

BBa_K2804004

CBD cipA fused to sfGFP

Composite

BBa_K2804005

CBD cipA fused to sfGFP under the control of LacI promoter

Composite

BBa_K2804006

Superfolder GFP with six-histidine tail

Coding

BBa_K2804014

CBD cipA fused to sfGFP

Composite

BBa_K2804015

CBD cipA fused to BMP2

Composite

 

Fusion protein to provide elasticity to the biomaterial
Coding parts
BBa_K2804011
ELP V-150
BBa_K2804012
ELP V-150
Composite part
BBa_K2804013
CBD cipA fused to ELP_C5 under the control of LacI promoter

Name

Description

Type

BBa_K2804011

ELP V-150

Coding

BBa_K2804012

ELP_C5

Coding

BBa_K2804013

CBD cipA fused to ELP_C5 under the control of LacI promoter

Composite

 

Cellulose production module
Coding parts
BBa_K2804008
CmcAx: Endo-beta-1,4-glucanase ezyme
BBa_K2804009
CcpAx: Cellulose-complementing protein
Composite part
BBa_K2804010
Operon member of bacterial cellulose synthase complex: Cmcax and CcpAx

Name

Description

Type

BBa_K2804008

CmcAx: Endo-beta-1,4-glucanase ezyme

Coding

BBa_K2804009

CcpAx: Cellulose-complementing protein

Coding

BBa_K2804010

Operon member of bacterial cellulose synthase complex: Cmcax and CcpAx

Composite
REFERENCES
Zhou, N., Li, Q., Lin, X. et al. Cell Tissue Res (2016) 366: 101. https://doi.org/10.1007/s00441-016-2403-0
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CONTACT US
biosin.espe@outlook.es

UNIVERSIDAD DE LAS FUERZAS ARMADAS ESPE.
Ecuador,Sangolquí, Av. General Rumiñahui s/n y Ambato