Difference between revisions of "Team:TU Darmstadt/Parts"

 
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            <div class="sidenav">
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                <a href="#Basic_Parts">Basic Parts</a>
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                <a href="#Composite_Parts">Composite Parts</a>
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                <a href="#Registry_Parts">Registry Parts</a>
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<h1><b> Basic Parts </b></h1>
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  <div class="col-lg-12 col-xl-8">
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<h1> Coding Sequences </h1>
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            <b>Due to COVID-19 </b> we were <b> not able to test </b> any of our designed parts in the lab. Nevertheless, we registered our basic parts, so <b> future teams </b> are easily able to characterize them. For our <b> Composite Parts we only uploaded one</b> for which we can provide modeling data. We refrained from uploading the others to the registry as there exists no primary literature on them, but we still provide schematic images on them. We did not submit any parts for the special prizes Best Basic Part and Best Composite Part. Parts we submit for the <b>medal criterion contribution</b> are also marked with <b>this symbol:</b> &hearts;.
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            <a  class="anchor" id="Basic_Parts"></a>
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<h2>Basic Parts</h2>
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<div class="headlinebar">
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                </div>
 
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<div class="TFcontainer">
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         <div class="containertext">
 
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<h2> CotA </h2>
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All <b>basic parts</b> we implemented into the design of our constructs are listed in the table below.
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<div class="containerimg">
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        </div>
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            <figure>
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    </div>
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                 <img src="https://static.igem.org/mediawiki/2020/d/d5/T--TU_Darmstadt--CD-ROM.jpg" alt="figure">
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<table class="table table-bordered">
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                 <figcaption id="Figure#">Figure 1: Rom-CD-Protein, host organism: Rom. </figcaption>
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  <thead>
 +
    <tr>
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      <th scope="col">Part(BBa_)</th>
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      <th scope="col">Type</th>
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      <th scope="col">Description</th>
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      <th scope="col">Length in bp</th>
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    </tr>
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  </thead>
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  <tbody>
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    <tr>
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      <td scope="row"><a href="http://parts.igem.org/Part:BBa_K3429000" target="_blank">BBa_K3429000</a></th>
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      <td>Promoter</td>
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      <td>P<sub><i>grac</sub></i> promoter</td>
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      <td>170</td>
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    </tr>
 +
    <tr>
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      <td scope="row"><a href="http://parts.igem.org/Part:BBa_K3429001" target="_blank">BBa_K3429001</a></th>
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      <td>Coding Sequence</td>
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      <td>TasA matrix protein</td>
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      <td>783</td>
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    </tr>
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<tr>
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      <td scope="row"><a href="http://parts.igem.org/Part:BBa_K3429002" target="_blank">BBa_K3429002</a></th>
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      <td>Coding Sequence</td>
 +
      <td>Superfolder Green Fluorescent Protein (sfGFP)</td>
 +
      <td>712</td>
 +
    </tr>
 +
<tr>
 +
      <td scope="row"><a href="http://parts.igem.org/Part:BBa_K3429003" target="_blank">BBa_K3429003</a></th>
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      <td>Coding Sequence</td>
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      <td>Protein Linker for fusion proteins: ARGGGGSGGGGSGS</td>
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      <td>42</td>
 +
    </tr>
 +
<tr>
 +
      <td scope="row"><a href="http://parts.igem.org/Part:BBa_K3429004" target="_blank">BBa_K3429004</a></th>
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      <td>Terminator</td>
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      <td><i>trpA</i> terminator</td>
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      <td>24</td>
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    </tr>
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<tr>
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      <td scope="row"><a href="http://parts.igem.org/Part:BBa_K3429005" target="_blank">BBa_K3429005</a></th>
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      <td>Coding Sequence</td>
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      <td>Ribosomal Protein S2 (rpsB)</i></td>
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      <td>721</td>
 +
    </tr>
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<tr>
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      <td scope="row"><a href="http://parts.igem.org/Part:BBa_K3429006" target="_blank">BBa_K3429006</a></th>
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      <td>Promoter</td>
 +
      <td>P<sub><i>degQ</sub></i> promoter</td>
 +
      <td>192</td>
 +
    </tr>
 +
<tr>
 +
      <td scope="row"><a href="http://parts.igem.org/Part:BBa_K3429007" target="_blank">BBa_K3429007</a></th>
 +
      <td>Terminator</td>
 +
      <td><i>degQ</i> terminator</td>
 +
      <td>36</td>
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    </tr>
 +
<tr>
 +
      <td scope="row"><a href="http://parts.igem.org/Part:BBa_K3429011" target="_blank">BBa_K3429011</a>&hearts;</th>
 +
      <td>Coding Sequence</td>
 +
      <td>Laccase CotA</td>
 +
      <td>1542</td>
 +
    </tr>
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<tr>
 +
      <td scope="row"><a href="http://parts.igem.org/Part:BBa_K3429012" target="_blank">BBa_K3429012</a>&hearts;</th>
 +
      <td>Coding Sequence</td>
 +
      <td>Blue copper oxidase CueO</td>
 +
      <td>1551</td>
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    </tr>
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 +
  </tbody>
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</table>
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            <a  class="anchor" id="Composite_Parts"></a>
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<h2>Composite Parts</h2>
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<div class="headlinebar">
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                </div>
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            <a  class="anchor" id="invitro_Characterization"></a>
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<h4> For <i>in vitro</i> Characterization </h4>
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<div class="TFcontainer">
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        <div class="containertext" id=Chapter1 >
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For purification and <a href="https://2020.igem.org/Team:TU_Darmstadt/Project/Pharmaceutical_Degradation#Experimentalapproach" target="_blank"><i>in vitro</i> characterization</a> of our enzymes we <b> designed the following Composite Parts </b>: <i>cotA</i>, <i> cueO</i>, and <i> ereB</i>. All of these Composite Parts contain a strep-tag for purification and are optimized for <I> E. coli</I>.  
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</div>
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</div>
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<br>
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<div class="containerimg" id=Chapter2>
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<figure>
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                 <a href="https://static.igem.org/mediawiki/2020/5/58/T--TU_Darmstadt--Composite_Pharmies.png" target="_blank"><img style="width:800px;" src="https://static.igem.org/mediawiki/2020/5/58/T--TU_Darmstadt--Composite_Pharmies.png" alt="figure"></a>
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                 <figcaption id="Figure#"><b>Figure 1:</b> Composite parts for <i>in &nbsp;vitro</i> characterization of our enzymes. Composite parts formed by T7 promoter, <i>lac</i> operator, the respective enzyme (including purification tag), and T7 terminator. Composite parts would have been introduced into the pET24(+) expression vector.</figcaption>
 
             </figure>
 
             </figure>
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</div>
 
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            <a  class="anchor" id="Immobilization"></a>       
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<h4> For Immobilization on our Biofilm </h4>
 
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<div class="TFcontainer">
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         <div class="containertext" id=Chapter1 >
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The following parts were designed for immobilization of our enzymes on the <a href="https://2020.igem.org/Team:TU_Darmstadt/Project/Biofilm#Displaying_Enzymes" target="_blank">TasA matrix protein</a> of our <i>B. subtilis </i> biofilm. In these composite parts the <b> enzymes are genetically fused to TasA</b>. We <a href="https://2020.igem.org/Team:TU_Darmstadt/Model/Enzyme_Modeling" target="_blank">modeled</a> the structure of the TasA-EreB fusion protein via MD simulation to see whether the enzyme remains stable (<a href="http://parts.igem.org/Part:BBa_K3429013" target="_blank">BBa_K3429013</a>&hearts;).
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<h2> CueO </h2>
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  </div>
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</div>
 
</div>
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</div>
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<br>
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<div class="containerimg" id=Chapter2>
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        <figure>
 +
        <a href="https://static.igem.org/mediawiki/2020/1/1d/T--TU_Darmstadt--Composite_Filmies.png" target="_blank"><img style="width:800px;" src="https://static.igem.org/mediawiki/2020/1/1d/T--TU_Darmstadt--Composite_Filmies.png"></a>
 +
                <figcaption id="Figure#"><b>Figure 2:</b> Composite parts for immobilization on biofilm matrix. TasA fusion proteins are flanked by the P<sub>grac</sub> promoter, <i>lacO</i> operator, and <i>trpA</i> terminator. Composite parts would have been introduced into the pSEVA3b67Rb shuttle vector.</figcaption>
 +
            </figure>
 +
        </div>
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            <a  class="anchor" id="Kill_Switch"></a>
 +
<h4> For our Kill Switch </h4>
 
<div class="TFcontainer">
 
<div class="TFcontainer">
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         <div class="containertext" id=Chapter1 >
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For our <a href="https://2020.igem.org/Team:TU_Darmstadt/Project/Kill_Switch#FinalKillSwitch" target="_blank">kill switch</a>, we combined already  <a href="#Registry_Parts">existing iGEM parts</a> with genetic elements of the <i> B. subtilis</i> <a href="https://2020.igem.org/Team:TU_Darmstadt/Background#Quorum%20Sensing" target="_blank">quorum sensing system</a>. Thus, generating a novel kill switch variant.
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<h2> EreB </h2>
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<br>
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<div class="containerimg" id=Chapter2>
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        <figure>
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        <a href="https://static.igem.org/mediawiki/2020/4/46/T--TU_Darmstadt--Switchies_Part.svg" target="_blank"><img style="width:800px;" src="https://static.igem.org/mediawiki/2020/4/46/T--TU_Darmstadt--Switchies_Part.svg"></a>
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                <figcaption id="Figure#"><b>Figure 3: </b> Composite parts for the kill switch. The essential gene rpsB is under control of the Pveg or PdegQ promoter. Because of the orientation of the lox 66 and lox 71 recombination sites, the sequence between them can be inverted by the Cre recombinase. The gene for the Cre recombinase is under control of the xylose induced PxylA promoter.</figcaption>
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            </figure>
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        </div>
  
 +
            <a  class="anchor" id="Registry_Parts"></a>
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<h2>Registry Parts</h2>
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<div class="headlinebar">
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                </div>
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<div class="TFcontainer">
 
<div class="TFcontainer">
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         <div class="containertext">For our project design we <b>took advantage of the iGEM registry</b> and took several parts from it. The parts are listed in the table below. For the well characterized part <a href="http://parts.igem.org/Part:BBa_K1159000" target="_blank">BBa_K1159000</a> from iGEM TU Munich 2013 we <b>generated the missing <a href="https://2020.igem.org/Team:TU_Darmstadt/Model/Enzyme_Modeling#EreB_CM" target="_blank">modelling data</a></b>. For <a href="http://parts.igem.org/Part:BBa_K1680007" target="_blank"> BBa_K1680007</a> we provide <b>further literature information</b>.  
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<h2> TasA </h2>
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Lorem ipsum dolor sit amet, consectetur adipiscing elit. Morbi feugiat ipsum ut lectus bibendum facilisis. Proin id dictum tortor. Mauris ac aliquet velit. Vestibulum a purus diam. Donec leo lorem, imperdiet id ante nec, rutrum blandit lacus. Phasellus in iaculis nulla. Cras scelerisque pellentesque lorem, at luctus ante volutpat a. Curabitur commodo porttitor sapien id ultrices. Proin rhoncus ut tortor id iaculis. Nulla enim diam, molestie at porttitor quis, vehicula non ante. In vitae scelerisque risus, et dignissim erat. Morbi dictum egestas porta. Phasellus quam mauris, fermentum sit amet magna ut, porttitor sodales quam. Pellentesque sed lectus augue. Orci varius natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Aenean ligula turpis, fermentum eget fermentum a, laoreet sed nibh.
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<h2> GFP </h2>
 
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<table class="table table-bordered">
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  <thead>
 +
    <tr>
 +
      <th scope="col">Part(BBa_)</th>
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      <th scope="col">Type</th>
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      <th scope="col">Description</th>
 +
      <th scope="col">Length in bp</th>
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    <th scope="col">Part created by</th>
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    </tr>
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  <tbody>
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<tr>
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<td scope="row"><a href=" http://parts.igem.org/Part:BBa_K733002" target="_blank"> BBa_K733002</a></th>
 +
      <td> Promoter </td>
 +
      <td> P<sub><i>XylA</sub></i> promoter </td>
 +
      <td>1387</td>
 +
      <td> iGEM HKUST Hong Kong 2012</td>
 +
    </tr>
 +
<tr>
 +
    <td scope="row"><a href="http://parts.igem.org/Part:BBa_K823003" target="_blank"> BBa_K823003</a></th>
 +
      <td> Promoter </td>
 +
      <td>P</i><sub>veg</sub></i> promoter</td>
 +
      <td>237</td>
 +
      <td> iGEM LMU Munich 2012</td>
 +
    </tr>
 +
<tr>
 +
      <td scope="row"><a href="http://parts.igem.org/Part:BBa_K1680007" target="_blank">BBa_K1680007</a>&hearts;</th>
 +
      <td>Coding Sequence</td>
 +
      <td>Cre recombinase</td>
 +
      <td>1029</td>
 +
      <td>iGEM Tuebingen 2015</td>
 +
    </tr>
 +
<tr>
 +
      <td scope="row"><a href="http://parts.igem.org/Part:BBa_I718016" target="_blank">BBa_I718016</a></th>
 +
      <td>Recombination Site</td>
 +
      <td>lox66 site</td>
 +
      <td>34</td>
 +
      <td>iGEM Paris 2007</td>
 +
    </tr>
 +
<tr>
 +
      <td scope="row"><a href="http://parts.igem.org/Part:BBa_I718017" target="_blank">BBa_I718017</a></th>
 +
      <td>Recombination Site</td>
 +
      <td>lox71 site</td>
 +
      <td>34</td>
 +
      <td>iGEM Paris 2007</td>
 +
    </tr>
 +
<tr>
 +
      <td scope="row"><a href="http://parts.igem.org/Part:BBa_K1159000" target="_blank">BBa_K1159000</a>&hearts; </th>
 +
      <td>Coding Sequence</td>
 +
      <td>Erythromycin Esterase Type II (EreB) in RFC[25]</td>
 +
      <td>1254</td>
 +
      <td>iGEM TU Munich 2013</td>
 +
    </tr>
 +
  </tbody>
 +
</table>
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{{TU_Darmstadt/Footer}}
 
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Latest revision as of 13:24, 26 October 2020

image/svg+xml - O O



Basic Parts Composite Parts Registry Parts

Due to COVID-19 we were not able to test any of our designed parts in the lab. Nevertheless, we registered our basic parts, so future teams are easily able to characterize them. For our Composite Parts we only uploaded one for which we can provide modeling data. We refrained from uploading the others to the registry as there exists no primary literature on them, but we still provide schematic images on them. We did not submit any parts for the special prizes Best Basic Part and Best Composite Part. Parts we submit for the medal criterion contribution are also marked with this symbol: ♥.

Basic Parts

All basic parts we implemented into the design of our constructs are listed in the table below.
Part(BBa_) Type Description Length in bp
BBa_K3429000 Promoter Pgrac promoter 170
BBa_K3429001 Coding Sequence TasA matrix protein 783
BBa_K3429002 Coding Sequence Superfolder Green Fluorescent Protein (sfGFP) 712
BBa_K3429003 Coding Sequence Protein Linker for fusion proteins: ARGGGGSGGGGSGS 42
BBa_K3429004 Terminator trpA terminator 24
BBa_K3429005 Coding Sequence Ribosomal Protein S2 (rpsB) 721
BBa_K3429006 Promoter PdegQ promoter 192
BBa_K3429007 Terminator degQ terminator 36
BBa_K3429011♥ Coding Sequence Laccase CotA 1542
BBa_K3429012♥ Coding Sequence Blue copper oxidase CueO 1551

Composite Parts

For in vitro Characterization

For purification and in vitro characterization of our enzymes we designed the following Composite Parts : cotA, cueO, and ereB. All of these Composite Parts contain a strep-tag for purification and are optimized for E. coli.

figure
Figure 1: Composite parts for in  vitro characterization of our enzymes. Composite parts formed by T7 promoter, lac operator, the respective enzyme (including purification tag), and T7 terminator. Composite parts would have been introduced into the pET24(+) expression vector.

For Immobilization on our Biofilm

The following parts were designed for immobilization of our enzymes on the TasA matrix protein of our B. subtilis biofilm. In these composite parts the enzymes are genetically fused to TasA. We modeled the structure of the TasA-EreB fusion protein via MD simulation to see whether the enzyme remains stable (BBa_K3429013♥).

Figure 2: Composite parts for immobilization on biofilm matrix. TasA fusion proteins are flanked by the Pgrac promoter, lacO operator, and trpA terminator. Composite parts would have been introduced into the pSEVA3b67Rb shuttle vector.

For our Kill Switch

For our kill switch, we combined already existing iGEM parts with genetic elements of the B. subtilis quorum sensing system. Thus, generating a novel kill switch variant.

Figure 3: Composite parts for the kill switch. The essential gene rpsB is under control of the Pveg or PdegQ promoter. Because of the orientation of the lox 66 and lox 71 recombination sites, the sequence between them can be inverted by the Cre recombinase. The gene for the Cre recombinase is under control of the xylose induced PxylA promoter.

Registry Parts


For our project design we took advantage of the iGEM registry and took several parts from it. The parts are listed in the table below. For the well characterized part BBa_K1159000 from iGEM TU Munich 2013 we generated the missing modelling data. For BBa_K1680007 we provide further literature information.
Part(BBa_) Type Description Length in bp Part created by
BBa_K733002 Promoter PXylA promoter 1387 iGEM HKUST Hong Kong 2012
BBa_K823003 Promoter Pveg promoter 237 iGEM LMU Munich 2012
BBa_K1680007♥ Coding Sequence Cre recombinase 1029 iGEM Tuebingen 2015
BBa_I718016 Recombination Site lox66 site 34 iGEM Paris 2007
BBa_I718017 Recombination Site lox71 site 34 iGEM Paris 2007
BBa_K1159000♥ Coding Sequence Erythromycin Esterase Type II (EreB) in RFC[25] 1254 iGEM TU Munich 2013