Difference between revisions of "Team:Wageningen UR/Composite Part"

 
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<h4><a href="#header">BioBricks</a></h4>
 
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<h1><b>BioBricks overview</b></h1>
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<h1><b>Composite parts</b></h1>
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<h2>Our best composite part: BBa_K1913011</h2>
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<p>
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<a href="http://parts.igem.org/Part:BBa_K1913011">BBa_K1913011</a> contains a vitamin b12 riboswitch with an inverter and reporter gene (mRFP). This Biobrick allows for inducible mRFP production based on the concentration of vitamin b12. Riboswitches are pieces of mRNA that can regulate gene expression depending on if it is bound to a ligand. They consists of two parts. An aptamer that binds a specific ligand, and an expression platform. This dual system makes riboswitches interesting tools for utilization within synthetic biology: aptamers and expression platform of different riboswitch sequences could potentially be combined to create new riboswitches. The riboswitch used for this biobrick is the <i>btuB</i> of <i>Escherichia coli</i> which normally stops expression after it binds to vitamin b12. Thanks to the TetR QPI system, expression of mRFP will start after binding to the ligand. <br>
 +
In our project, the vitamin b12 riboswitch is used to <a href="https://2016.igem.org/Team:Wageningen_UR/Description/Regulation#DetectingMites">detect <i>Varroa</i> mites</a> in beehives. The mites feed on the bees’ hemolymph, of which vitamin b12 is a component. BeeT can thus sense whether bees have been bitten, and respond by producing an anti-<i>Varroa</i> toxin. <br>
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</p>
  
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<p>Below, you can find all the biobricks we created!</p>
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<p><br>
 +
<b>Below, you can find all composite parts we created!</b></p>
  
 
<table>
 
<table>
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     <th>BioBrick name</th>     
 
     <th>BioBrick name</th>     
 
     <th>Designer</th>
 
     <th>Designer</th>
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  </tr>
 
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  <tr>
 
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    <td><a href="http://parts.igem.org/Part:BBa_K1913000">BBa_K1913000</a></td>
 
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    <td>chiA for Varroa destructor</td>
 
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    <td>Lisa</td>
 
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  </tr>
 
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  <tr>
 
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    <td><a href="http://parts.igem.org/Part:BBa_K1913001">BBa_K1913001</a></td>
 
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    <td>chiB for Varroa destructor</td>
 
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    <td>Lisa</td>
 
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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     <td><a href="http://parts.igem.org/Part:BBa_K19130014">BBa_K19130014</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913014">BBa_K1913014</a></td>
 
     <td>3-oxo-hexanoyl-HSL GFP reporter</td>
 
     <td>3-oxo-hexanoyl-HSL GFP reporter</td>
 
     <td>Thomas</td>
 
     <td>Thomas</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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     <td><a href="http://parts.igem.org/Part:BBa_K19130016">BBa_K19130016</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913016">BBa_K1913016</a></td>
 
     <td>434- and lambda cI balance RFP reporter</td>
 
     <td>434- and lambda cI balance RFP reporter</td>
 
     <td>Thomas</td>
 
     <td>Thomas</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913008">BBa_K1913008</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913010">BBa_K1913010</a></td>
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    <td>vitamin b12 riboswitch</td>
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    <td>Carina</td>
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  </tr>
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  <tr>
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    <td><a href="http://parts.igem.org/Part:BBa_K1913009">BBa_K1913009</a></td>
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    <td>Guanine riboswitch</td>
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    <td>Carina</td>
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  </tr>
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  <tr>
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    <td><a href="http://parts.igem.org/Part:BBa_K19130010">BBa_K19130010</a></td>
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     <td>tetR QPI + mRFP</td>
 
     <td>tetR QPI + mRFP</td>
 
     <td>Carina</td>
 
     <td>Carina</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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     <td><a href="http://parts.igem.org/Part:BBa_K19130011">BBa_K19130011</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913011">BBa_K1913011</a></td>
 
     <td>Vitamin b12 riboswitch + mRFP</td>
 
     <td>Vitamin b12 riboswitch + mRFP</td>
 
     <td>Carina</td>
 
     <td>Carina</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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     <td><a href="http://parts.igem.org/Part:BBa_K19130012">BBa_K19130012</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913012">BBa_K1913012</a></td>
 
     <td>Guanine riboswitch + guanine riboswitch</td>
 
     <td>Guanine riboswitch + guanine riboswitch</td>
 
     <td>Carina</td>
 
     <td>Carina</td>
 
   </tr>
 
   </tr>
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  <tr>
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<tr>
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     <td><a href="http://parts.igem.org/Part:BBa_K19130019">BBa_K19130019</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913027">BBa_K1913027</a></td>
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    <td>Guanine riboswitch BS-yxjA</td>
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    <td>Tianhe</td>
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  </tr>
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  <tr>
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    <td><a href="http://parts.igem.org/Part:BBa_K19130020">BBa_K19130020</a></td>
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    <td>mRFP with degredation tag</td>
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    <td>Tianhe</td>
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  </tr>
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  <tr>
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    <td><a href="http://parts.igem.org/Part:BBa_K19130021">BBa_K19130021</a></td>
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    <td>sGFP with defredation tag</td>
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    <td>Tianhe</td>
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  </tr>
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  <tr>
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    <td><a href="http://parts.igem.org/Part:BBa_K19130022">BBa_K19130022</a></td>
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    <td>Artificial FixK2 promoter with lac operon O1, O3 consitutive promoter </td>
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    <td>Tianhe</td>
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  </tr>
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  <tr>
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    <td><a href="http://parts.igem.org/Part:BBa_K19130023">BBa_K19130023</a></td>
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    <td>Artificial FixK2 promoter with lac operon O1, O3 ompR</td>
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    <td>Tianhe</td>
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  </tr>
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  <tr>
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    <td><a href="http://parts.igem.org/Part:BBa_K19130024">BBa_K19130024</a></td>
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    <td>Artificial FixK2 promoter with two tetO operons</td>
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    <td>Tianhe</td>
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  </tr>
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  <tr>
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    <td><a href="http://parts.igem.org/Part:BBa_K19130025">BBa_K19130025</a></td>
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    <td>Natural FixK2 promoter with lac operons O1, O3</td>
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    <td>Tianhe</td>
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  </tr>
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  <tr>
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    <td><a href="http://parts.igem.org/Part:BBa_K19130026">BBa_K19130026</a></td>
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    <td>Natural FixK2 promoter with two tetO operons</td>
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    <td>Tianhe</td>
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−
  </tr>
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  <tr>
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    <td><a href="http://parts.igem.org/Part:BBa_K19130027">BBa_K19130027</a></td>
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     <td>Wild type plac-FixK2 hybrid promoter with mRFP</td>
 
     <td>Wild type plac-FixK2 hybrid promoter with mRFP</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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     <td><a href="http://parts.igem.org/Part:BBa_K19130028">BBa_K19130028</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913028">BBa_K1913028</a></td>
 
     <td>Wild type ptet-FixK2 hybrid promoter with mRFP</td>
 
     <td>Wild type ptet-FixK2 hybrid promoter with mRFP</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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     <td><a href="http://parts.igem.org/Part:BBa_K19130029">BBa_K19130029</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913029">BBa_K1913029</a></td>
 
     <td>Synthetic plac-FixK2 hybrid promoter +RBS with mRFP</td>
 
     <td>Synthetic plac-FixK2 hybrid promoter +RBS with mRFP</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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     <td><a href="http://parts.igem.org/Part:BBa_K19130030">BBa_K19130030</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913030">BBa_K1913030</a></td>
 
     <td>Synthetic plac-FixK2 hybrid promoter+RBS with mRFP</td>
 
     <td>Synthetic plac-FixK2 hybrid promoter+RBS with mRFP</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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     <td><a href="http://parts.igem.org/Part:BBa_K19130031">BBa_K19130031</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913031">BBa_K1913031</a></td>
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     <td>Syntheitc ptet-FixK2 hybrid promoter+RBS with mRFP</td>
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     <td>Synthetic ptet-FixK2 hybrid promoter+RBS with mRFP</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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     <td><a href="http://parts.igem.org/Part:BBa_K19130032">BBa_K19130032</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913032">BBa_K1913032</a></td>
 
     <td>Toggle Switch device</td>
 
     <td>Toggle Switch device</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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     <td><a href="http://parts.igem.org/Part:BBa_K19130033">BBa_K19130033</a></td>
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     <td><a href="http://parts.igem.org/Part:BBa_K1913033">BBa_K1913033</a></td>
 
     <td>Toggle Switch device</td>
 
     <td>Toggle Switch device</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
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  </tr>
 
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  <tr>
 
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    <td><a href="http://parts.igem.org/Part:BBa_K19130013">BBa_K19130013</a></td>
 
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    <td>Cry3Aa + RBS + TEV + His-tag</td>
 
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    <td>Jaccoline/Linea</td>
 
 
   </tr>
 
   </tr>
 
</table>
 
</table>
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<p>
 
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Besides, we submitted one part that cannot be classified as a biobrick because it has some illegal restriction sites:</p>
 
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<table> 
 
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  <tr>
 
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    <th>BioBrick number</th>
 
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    <th>BioBrick name</th>     
 
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    <th>Designer</th>
 
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  </tr>
 
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<tr>
 
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    <td><a href="http://parts.igem.org/Part:BBa_K19130015">BBa_K19130015</a></td>
 
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    <td>Cry3Aa with araC/pBAD</td>
 
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    <td>Jaccoline/Linea</td>
 
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  </tr>
 
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</table>
 
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<p>
 
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Initally, part BBa_K19130015 was made only to for testing the <a href="https://2016.igem.org/Team:Wageningen_UR/Description/Specificity#Assay"><i>in vitro</i> assay</a>, <b>jacco, please put the rest of your excuse here ;)</b>.
 
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</p>
 
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<p>
 
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<b>No Cas9 biobrick?!</b><br>
 
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When we started making the constructs for the <a href="https://2016.igem.org/Team:Wageningen_UR/Description/Biocontainment#SAA">Cas9 kill switch</a>, two approaches were taken: one was taking the Cas9 that is available in the iGEM registry (BBa_K1218011) as a starting point for making mutations and expressing Cas9. The other approach was starting with pdCas9 (Addgene plasmid # 46569). While cloning, it proved to be difficult to transfer BBa_K1218011 to another backbone (see also the <a href="https://2016.igem.org/Team:Wageningen_UR/Notebook/Cas9">notebook</a>). Furthermore, when cultures transformed with BBa_K1218011 were checked for Cas9 expression with SDS-PAGE, no convincing Cas9 band could be observed. Because of time limitations we decided to continue working with the Addgene construct for making the mutations, and chose an established system for protein expression. For that reason, no Cas9-biobricks were submitted. Furthermore, the pEVOL construct containing an aminoacyl-synthetase and a tRNA for introducing BipA in response to the TAG stopcodon were isolated from a strain kindly received from George Church (described in <a href="http://www.nature.com/nature/journal/v518/n7537/full/nature14121.html">Mandel <i>et al.</i>, 2015</a>). The MTA that was signed to receive the strain does not allow for redistribution. 
 
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Latest revision as of 09:10, 17 October 2016

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Wageningen UR iGEM 2016

 

 

 

Composite parts

Our best composite part: BBa_K1913011

BBa_K1913011 contains a vitamin b12 riboswitch with an inverter and reporter gene (mRFP). This Biobrick allows for inducible mRFP production based on the concentration of vitamin b12. Riboswitches are pieces of mRNA that can regulate gene expression depending on if it is bound to a ligand. They consists of two parts. An aptamer that binds a specific ligand, and an expression platform. This dual system makes riboswitches interesting tools for utilization within synthetic biology: aptamers and expression platform of different riboswitch sequences could potentially be combined to create new riboswitches. The riboswitch used for this biobrick is the btuB of Escherichia coli which normally stops expression after it binds to vitamin b12. Thanks to the TetR QPI system, expression of mRFP will start after binding to the ligand.
In our project, the vitamin b12 riboswitch is used to detect Varroa mites in beehives. The mites feed on the bees’ hemolymph, of which vitamin b12 is a component. BeeT can thus sense whether bees have been bitten, and respond by producing an anti-Varroa toxin.


Below, you can find all composite parts we created!

BioBrick number BioBrick name Designer
BBa_K1913002 chiA device regulated by pBAD Lisa
BBa_K1913003 chiB device regulated by pBAD Lisa
BBa_K1913005 lux quorum sensing system + GFP reporter Thomas
BBa_K1913006 434- and lambda cI balance operon + mRFP reporter Thomas
BBa_K1913007 434- and lambda cI operon for tuning protein balance Thomas
BBa_K1913014 3-oxo-hexanoyl-HSL GFP reporter Thomas
BBa_K1913016 434- and lambda cI balance RFP reporter Thomas
BBa_K1913010 tetR QPI + mRFP Carina
BBa_K1913011 Vitamin b12 riboswitch + mRFP Carina
BBa_K1913012 Guanine riboswitch + guanine riboswitch Carina
BBa_K1913027 Wild type plac-FixK2 hybrid promoter with mRFP Tianhe
BBa_K1913028 Wild type ptet-FixK2 hybrid promoter with mRFP Tianhe
BBa_K1913029 Synthetic plac-FixK2 hybrid promoter +RBS with mRFP Tianhe
BBa_K1913030 Synthetic plac-FixK2 hybrid promoter+RBS with mRFP Tianhe
BBa_K1913031 Synthetic ptet-FixK2 hybrid promoter+RBS with mRFP Tianhe
BBa_K1913032 Toggle Switch device Tianhe
BBa_K1913033 Toggle Switch device Tianhe