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

 
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<h4><a href="#header">BioBricks</a></h4>
 
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<h1><b>BioBricks overview</b></h1>
+
<h1><b>Basic parts</b></h1>
 +
 
 +
<h2>Our best basic part: BBa_K1913025</h2>
 +
<br>
 +
 
 +
<p>
 +
<a href="http://parts.igem.org/Part:BBa_K1913025">BBa_K1913025</a> encodes the plac-FixK2 hybrid promoter. The sequence of upper control element and core element region are derived from the wildtype FixK2 promoter from the bacterium <i>Bradyrhizobium japonicum</i>. <br>
 +
BBa_K1913025 can be be activated by phosphorylated FixJ, part of the YF1-FixJ light sensor. In the presence of light, FixJ is no longer phosphorylated and BBa_K1913025 is not activated. At the same time, it can be inhibited by the LacI repressor because of two lac operators that are positioned on both sides of the FixK2 promoter core. <br>
 +
The BBa_K1913025 was tested together with the YF1-FixJ light sensor using mRFP as a reporter. After culturing for 24 hours in darkness, BBa_K1913025 has five times higher fluorescence than a control without the light sensor. <br>
 +
In our project, the plac-FixK2 promoter is part of the <a href="https://2016.igem.org/Team:Wageningen_UR/Description/Regulation#Toggleswitch">toggle switch</a>. This switch is intended for regulating Bt toxin expression. The promoter is only activated in darkness, inside the beehive. Furthermore, it stays inactivated through LacI mediated repression until B12 is sensed, which inhibits the formation of LacI (see also our project about <a href="https://2016.igem.org/Team:Wageningen_UR/Description/Regulation#DetectingMites">detecting <i>Varroa</i> mites</a>)
 +
</p>
  
<p>Below, you can find all the biobricks we created!</p>
+
<p><br>
 +
<b>Below, you can find all basic parts we created!</b></p>
  
 
<table>
 
<table>
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     <td>chiB for Varroa destructor</td>
 
     <td>chiB for Varroa destructor</td>
 
     <td>Lisa</td>
 
     <td>Lisa</td>
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K1913002">BBa_K1913002</a></td>
 
    <td>chiA device regulated by pBAD</td>
 
    <td>Lisa</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K1913003">BBa_K1913003</a></td>
 
    <td>chiB device regulated by pBAD</td>
 
    <td>Lisa</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K1913005">BBa_K1913005</a></td>
 
    <td>lux quorum sensing system + GFP reporter</td>
 
    <td>Thomas</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K1913006">BBa_K1913006</a></td>
 
    <td>434- and lambda cI balance operon + mRFP reporter</td>
 
    <td>Thomas</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K1913007">BBa_K1913007</a></td>
 
    <td>434- and lambda cI operon for tuning protein balance</td>
 
    <td>Thomas</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K19130014">BBa_K19130014</a></td>
 
    <td>3-oxo-hexanoyl-HSL GFP reporter</td>
 
    <td>Thomas</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K19130016">BBa_K19130016</a></td>
 
    <td>434- and lambda cI balance RFP reporter</td>
 
    <td>Thomas</td>
 
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
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   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td><a href="http://parts.igem.org/Part:BBa_K19130010">BBa_K19130010</a></td>
+
     <td><a href="http://parts.igem.org/Part:BBa_K1913019">BBa_K1913019</a></td>
    <td>tetR QPI + mRFP</td>
+
    <td>Carina</td>
+
  </tr>
+
  <tr>
+
    <td><a href="http://parts.igem.org/Part:BBa_K19130011">BBa_K19130011</a></td>
+
    <td>Vitamin b12 riboswitch + mRFP</td>
+
    <td>Carina</td>
+
  </tr>
+
  <tr>
+
    <td><a href="http://parts.igem.org/Part:BBa_K19130012">BBa_K19130012</a></td>
+
    <td>Guanine riboswitch + guanine riboswitch</td>
+
    <td>Carina</td>
+
  </tr>
+
  <tr>
+
    <td><a href="http://parts.igem.org/Part:BBa_K19130019">BBa_K19130019</a></td>
+
 
     <td>Guanine riboswitch BS-yxjA</td>
 
     <td>Guanine riboswitch BS-yxjA</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td><a href="http://parts.igem.org/Part:BBa_K19130020">BBa_K19130020</a></td>
+
     <td><a href="http://parts.igem.org/Part:BBa_K1913020">BBa_K1913020</a></td>
 
     <td>mRFP with degredation tag</td>
 
     <td>mRFP with degredation tag</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td><a href="http://parts.igem.org/Part:BBa_K19130021">BBa_K19130021</a></td>
+
     <td><a href="http://parts.igem.org/Part:BBa_K1913021">BBa_K1913021</a></td>
 
     <td>sGFP with defredation tag</td>
 
     <td>sGFP with defredation tag</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td><a href="http://parts.igem.org/Part:BBa_K19130022">BBa_K19130022</a></td>
+
     <td><a href="http://parts.igem.org/Part:BBa_K1913022">BBa_K1913022</a></td>
     <td>Artificial FixK2 promoter with lac operon O1, O3 consitutive promoter </td>
+
     <td>Synthetic plac-FixK2 hybrid promoter with RBS</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td><a href="http://parts.igem.org/Part:BBa_K19130023">BBa_K19130023</a></td>
+
     <td><a href="http://parts.igem.org/Part:BBa_K1913023">BBa_K1913023</a></td>
     <td>Artificial FixK2 promoter with lac operon O1, O3 ompR</td>
+
     <td>Synthetic plac-FixK2 hybrid promoter with RBS</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td><a href="http://parts.igem.org/Part:BBa_K19130024">BBa_K19130024</a></td>
+
     <td><a href="http://parts.igem.org/Part:BBa_K1913024">BBa_K1913024</a></td>
     <td>Artificial FixK2 promoter with two tetO operons</td>
+
     <td>Synthetic ptet-FixK2 hybrid promoter with RBS</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td><a href="http://parts.igem.org/Part:BBa_K19130025">BBa_K19130025</a></td>
+
     <td><a href="http://parts.igem.org/Part:BBa_K1913025">BBa_K1913025</a></td>
     <td>Natural FixK2 promoter with lac operons O1, O3</td>
+
     <td>Wild type plac-FixK2 hybrid promoter</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td><a href="http://parts.igem.org/Part:BBa_K19130026">BBa_K19130026</a></td>
+
     <td><a href="http://parts.igem.org/Part:BBa_K1913026">BBa_K1913026</a></td>
     <td>Natural FixK2 promoter with two tetO operons</td>
+
     <td>Wild type ptet-FixK2 hybrid promoter</td>
 
     <td>Tianhe</td>
 
     <td>Tianhe</td>
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K19130027">BBa_K19130027</a></td>
 
    <td>Wild type plac-FixK2 hybrid promoter with mRFP</td>
 
    <td>Tianhe</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K19130028">BBa_K19130028</a></td>
 
    <td>Wild type ptet-FixK2 hybrid promoter with mRFP</td>
 
    <td>Tianhe</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K19130029">BBa_K19130029</a></td>
 
    <td>Synthetic plac-FixK2 hybrid promoter +RBS with mRFP</td>
 
    <td>Tianhe</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K19130030">BBa_K19130030</a></td>
 
    <td>Synthetic plac-FixK2 hybrid promoter+RBS with mRFP</td>
 
    <td>Tianhe</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K19130031">BBa_K19130031</a></td>
 
    <td>Syntheitc ptet-FixK2 hybrid promoter+RBS with mRFP</td>
 
    <td>Tianhe</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K19130032">BBa_K19130032</a></td>
 
    <td>Toggle Switch device</td>
 
    <td>Tianhe</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K19130033">BBa_K19130033</a></td>
 
    <td>Toggle Switch device</td>
 
    <td>Tianhe</td>
 
  </tr>
 
  <tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K19130013">BBa_K19130013</a></td>
 
    <td>Cry3Aa + RBS + TEV + His-tag</td>
 
    <td>Jaccoline/Linea</td>
 
 
   </tr>
 
   </tr>
 
</table>
 
</table>
  
<p>
 
Besides, we submitted one part that cannot be classified as a biobrick because it has some illegal restriction sites:</p>
 
  
<table> 
 
  <tr>
 
    <th>BioBrick number</th>
 
    <th>BioBrick name</th>     
 
    <th>Designer</th>
 
  </tr>
 
<tr>
 
    <td><a href="http://parts.igem.org/Part:BBa_K19130015">BBa_K19130015</a></td>
 
    <td>Cry3Aa with araC/pBAD</td>
 
    <td>Jaccoline/Linea</td>
 
  </tr>
 
</table>
 
 
<p>
 
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>.
 
</p>
 
 
<p>
 
<b>No Cas9 biobrick?!</b><br>
 
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. 
 
</p>
 
  
  
 
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Latest revision as of 09:43, 17 October 2016

Wageningen UR iGEM 2016

 

 

Basic parts

Our best basic part: BBa_K1913025


BBa_K1913025 encodes the plac-FixK2 hybrid promoter. The sequence of upper control element and core element region are derived from the wildtype FixK2 promoter from the bacterium Bradyrhizobium japonicum.
BBa_K1913025 can be be activated by phosphorylated FixJ, part of the YF1-FixJ light sensor. In the presence of light, FixJ is no longer phosphorylated and BBa_K1913025 is not activated. At the same time, it can be inhibited by the LacI repressor because of two lac operators that are positioned on both sides of the FixK2 promoter core.
The BBa_K1913025 was tested together with the YF1-FixJ light sensor using mRFP as a reporter. After culturing for 24 hours in darkness, BBa_K1913025 has five times higher fluorescence than a control without the light sensor.
In our project, the plac-FixK2 promoter is part of the toggle switch. This switch is intended for regulating Bt toxin expression. The promoter is only activated in darkness, inside the beehive. Furthermore, it stays inactivated through LacI mediated repression until B12 is sensed, which inhibits the formation of LacI (see also our project about detecting Varroa mites)


Below, you can find all basic parts we created!

BioBrick number BioBrick name Designer
BBa_K1913000 chiA for Varroa destructor Lisa
BBa_K1913001 chiB for Varroa destructor Lisa
BBa_K1913008 vitamin b12 riboswitch Carina
BBa_K1913009 Guanine riboswitch Carina
BBa_K1913019 Guanine riboswitch BS-yxjA Tianhe
BBa_K1913020 mRFP with degredation tag Tianhe
BBa_K1913021 sGFP with defredation tag Tianhe
BBa_K1913022 Synthetic plac-FixK2 hybrid promoter with RBS Tianhe
BBa_K1913023 Synthetic plac-FixK2 hybrid promoter with RBS Tianhe
BBa_K1913024 Synthetic ptet-FixK2 hybrid promoter with RBS Tianhe
BBa_K1913025 Wild type plac-FixK2 hybrid promoter Tianhe
BBa_K1913026 Wild type ptet-FixK2 hybrid promoter Tianhe