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<path fill-rule="evenodd" d="M1.646 2.646a.5.5 0 0 1 .708 0L8 8.293l5.646-5.647a.5.5 0 0 1 .708.708l-6 6a.5.5 0 0 1-.708 0l-6-6a.5.5 0 0 1 0-.708z"/> | <path fill-rule="evenodd" d="M1.646 2.646a.5.5 0 0 1 .708 0L8 8.293l5.646-5.647a.5.5 0 0 1 .708.708l-6 6a.5.5 0 0 1-.708 0l-6-6a.5.5 0 0 1 0-.708z"/> | ||
− | </svg> | + | </svg> <span id="1"></span> |
</div> | </div> | ||
</div> | </div> | ||
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<ul class="dropdown-menu dropdown-menu-end fade-down" aria-labelledby="navbarDropdown"> | <ul class="dropdown-menu dropdown-menu-end fade-down" aria-labelledby="navbarDropdown"> | ||
<li><a class="dropdown-item" href="https://2021.igem.org/Team:IISER-Tirupati_India/Team">MEMBERS</a></li> | <li><a class="dropdown-item" href="https://2021.igem.org/Team:IISER-Tirupati_India/Team">MEMBERS</a></li> | ||
− | <li><a class="dropdown-item" href="https://2021.igem.org/Team:IISER-Tirupati_India/Attributions"> | + | <li><a class="dropdown-item" href="https://2021.igem.org/Team:IISER-Tirupati_India/Attributions">ATTRIBUTIONS</a></li> |
<li><a class="dropdown-item" href="https://2021.igem.org/Team:IISER-Tirupati_India/Collaborations">COLLABORATIONS </a></li> | <li><a class="dropdown-item" href="https://2021.igem.org/Team:IISER-Tirupati_India/Collaborations">COLLABORATIONS </a></li> | ||
<li><a class="dropdown-item" href="https://2021.igem.org/Team:IISER-Tirupati_India/Partnership">PARTNERSHIP</a></li> | <li><a class="dropdown-item" href="https://2021.igem.org/Team:IISER-Tirupati_India/Partnership">PARTNERSHIP</a></li> | ||
</ul> | </ul> | ||
+ | </li> | ||
+ | <li class="nav-item dropdown"> | ||
+ | <a class="nav-link active" href="https://2021.igem.org/Team:IISER-Tirupati_India/Awards" role="button"> | ||
+ | AWARDS | ||
+ | </a> | ||
</li> | </li> | ||
</ul> | </ul> | ||
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<li><a class="index_link" href="#4">Composite Parts</a></li> | <li><a class="index_link" href="#4">Composite Parts</a></li> | ||
<li><a class="index_link" href="#5">Improvement</a></li> | <li><a class="index_link" href="#5">Improvement</a></li> | ||
+ | <li><a class="index_link" href="#6">References</a></li> | ||
</ul> | </ul> | ||
</section> | </section> | ||
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<div class="col-md-8 px-md-5"> | <div class="col-md-8 px-md-5"> | ||
<div> | <div> | ||
− | <p class="py-3 | + | <p class="py-3"> |
</p> | </p> | ||
<h2>Overview</h2> | <h2>Overview</h2> | ||
− | <p>Due to the novel approaches taken while pursuing this project, we found ourselves using new parts that had not been given in the iGEM registry. In total, we developed 37 new basic parts (either entirely new or by modifying previously existing ones). We strongly believe that these two collections can be exploited to their full potential by the subsequent iGEM generations.</p> | + | <p>Due to the novel approaches taken while pursuing this project, we found ourselves using new parts that had not been given in the iGEM registry. In total, we developed <strong>37 new basic parts</strong> (either entirely new or by modifying previously existing ones). We strongly believe that these two collections can be exploited to their full potential by the subsequent iGEM generations.</p> |
<p id="2">Furthermore, we have designed and used multiple combinations of basic parts as given in the composite part section.These composite parts can be used in multiple settings depending on the requirements of the projects.</p> | <p id="2">Furthermore, we have designed and used multiple combinations of basic parts as given in the composite part section.These composite parts can be used in multiple settings depending on the requirements of the projects.</p> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K316001">BBa_K316001</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K316001" target="_blank" style="color:#0645AD;">BBa_K316001</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K143013">BBa_K143013</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K143013" target="_blank" style="color:#0645AD;">BBa_K143013</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K143014">BBa_K143014</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K143014" target="_blank" style="color:#0645AD;">BBa_K143014</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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</td> | </td> | ||
</tr> | </tr> | ||
+ | |||
<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part: | + | <p><a href="http://parts.igem.org/Part:BBa_K2333002" target="_blank" style="color:#0645AD;">BBa_K2333002</a></p> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K1351031"> | + | <p><a href="http://parts.igem.org/Part:BBa_K1351031" target="_blank" style="color:#0645AD;">BBa_K1351028</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K1351030">BBa_K1351030</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K1351030" target="_blank" style="color:#0645AD;">BBa_K1351030</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K1351031">BBa_K1351031</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K1351031" target="_blank" style="color:#0645AD;">BBa_K1351031</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K1351033">BBa_K1351033</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K1351033" target="_blank" style="color:#0645AD;">BBa_K1351033</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_J06504">BBa_J06504</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_J06504" target="_blank" style="color:#0645AD;">BBa_J06504</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_B0015" target="_blank" style="color:#0645AD;">BBa_B0015</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="https://parts.igem.org/Part:BBa_B0010">BBa_B0010</a></p> | + | <p><a href="https://parts.igem.org/Part:BBa_B0010" target="_blank" style="color:#0645AD;">BBa_B0010</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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</td> | </td> | ||
<td> | <td> | ||
− | <p | + | <p>Relatively weaker Terminator as compared to <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank" style="color:#0645AD;">BBa_B0015</a></p> |
</td> | </td> | ||
</tr> | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p id="3"><a href="http://parts.igem.org/Part:BBa_K2314608" target="_blank" style="color:#0645AD;">BBa_K2314608</a></p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p>T<sub>Mini</sub></p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p>68 bp</p> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p>Very short terminator in yeast with good performance </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889000">BBa_K3889000</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889000" target="_blank" style="color:#0645AD;">BBa_K3889000</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889002">BBa_K3889002</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889002" target="_blank" style="color:#0645AD;">BBa_K3889002</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889010">BBa_K3889010</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889010" target="_blank" style="color:#0645AD;">BBa_K3889010</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889011">BBa_K3889011</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889011" target="_blank" style="color:#0645AD;">BBa_K3889011</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889012">BBa_K3889012</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889012" target="_blank" style="color:#0645AD;">BBa_K3889012</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889013">BBa_K3889013</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889013" target="_blank" style="color:#0645AD;">BBa_K3889013</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889014">BBa_K3889014</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889014" target="_blank" style="color:#0645AD;">BBa_K3889014</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889020">BBa_K3889020</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889020" target="_blank" style="color:#0645AD;">BBa_K3889020</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889021">BBa_K3889021</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889021" target="_blank" style="color:#0645AD;">BBa_K3889021</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889022">BBa_K3889022</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889022" target="_blank" style="color:#0645AD;">BBa_K3889022</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889023">BBa_K3889023</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889023" target="_blank" style="color:#0645AD;">BBa_K3889023</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889024">BBa_K3889024</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889024" target="_blank" style="color:#0645AD;">BBa_K3889024</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889025">BBa_K3889025</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889025" target="_blank" style="color:#0645AD;">BBa_K3889025</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p>yqcG toxin | + | <p>yqcG toxin</p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889026">BBa_K3889026</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889026" target="_blank" style="color:#0645AD;">BBa_K3889026</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p>ytvA light sensor | + | <p>ytvA light sensor</p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889027">BBa_K3889027</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889027" target="_blank" style="color:#0645AD;">BBa_K3889027</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889028">BBa_K3889028</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889028" target="_blank" style="color:#0645AD;">BBa_K3889028</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889029">BBa_K3889029</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889029" target="_blank" style="color:#0645AD;">BBa_K3889029</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889030">BBa_K3889030</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889030" target="_blank" style="color:#0645AD;">BBa_K3889030</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889040">BBa_K3889040</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889040" target="_blank" style="color:#0645AD;">BBa_K3889040</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889050">BBa_K3889050</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889050" target="_blank" style="color:#0645AD;">BBa_K3889050</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889051">BBa_K3889051</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889051" target="_blank" style="color:#0645AD;">BBa_K3889051</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889069">BBa_K3889069</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889069" target="_blank" style="color:#0645AD;">BBa_K3889069</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889070">BBa_K3889070</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889070" target="_blank" style="color:#0645AD;">BBa_K3889070</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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</td> | </td> | ||
<td> | <td> | ||
− | <p>Fused product of nagP and <a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> (Improvement in <a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a>)</p> | + | <p>Fused product of nagP and <a href="http://parts.igem.org/Part:BBa_B0010" target="_blank" style="color:#0645AD;">BBa_B0010</a> (Improvement in <a href="http://parts.igem.org/Part:BBa_B0010" target="_blank" style="color:#0645AD;">BBa_B0010</a>)</p> |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889080">BBa_K3889080</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889080" target="_blank" style="color:#0645AD;">BBa_K3889080</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889081">BBa_K3889081</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889081" target="_blank" style="color:#0645AD;">BBa_K3889081</a></p> |
</td> | </td> | ||
<td> | <td> | ||
Line 707: | Line 715: | ||
<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889082">BBa_K3889082</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889082" target="_blank" style="color:#0645AD;">BBa_K3889082</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889083">BBa_K3889083</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889083" target="_blank" style="color:#0645AD;">BBa_K3889083</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889084">BBa_K3889084</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889084" target="_blank" style="color:#0645AD;">BBa_K3889084</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889085">BBa_K3889085</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889085" target="_blank" style="color:#0645AD;">BBa_K3889085</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889086">BBa_K3889086</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889086" target="_blank" style="color:#0645AD;">BBa_K3889086</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889087">BBa_K3889087</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889087" target="_blank" style="color:#0645AD;">BBa_K3889087</a></p> |
</td> | </td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889088">BBa_K3889088</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889088" target="_blank" style="color:#0645AD;">BBa_K3889088</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889089">BBa_K3889089</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889089" target="_blank" style="color:#0645AD;">BBa_K3889089</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889090">BBa_K3889090</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889090" target="_blank" style="color:#0645AD;">BBa_K3889090</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889091">BBa_K3889091</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889091" target="_blank" style="color:#0645AD;">BBa_K3889091</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889092">BBa_K3889092</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889092" target="_blank" style="color:#0645AD;">BBa_K3889092</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889093">BBa_K3889093</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889093" target="_blank" style="color:#0645AD;">BBa_K3889093</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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</td> | </td> | ||
<td> | <td> | ||
− | < | + | <span id="4">Anti-toxin of yqcG (a kind of DNase)</span> |
</td> | </td> | ||
</tr> | </tr> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889100">BBa_K3889100</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889100" target="_blank" style="color:#0645AD;">BBa_K3889100</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p>SRTF1 | + | <p>SRTF1 expression cassette</p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889101">BBa_K3889101</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889101" target="_blank" style="color:#0645AD;">BBa_K3889101</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p>P22 | + | <p>P22 expression cassette</p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889102">BBa_K3889102</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889102" target="_blank" style="color:#0645AD;">BBa_K3889102</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p>Ovastacin | + | <p>Ovastacin expression cassette</p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889110">BBa_K3889110</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889110" target="_blank" style="color:#0645AD;">BBa_K3889110</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p> | + | <p>XylR Repressor Cassette</p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889111">BBa_K3889111</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889111" target="_blank" style="color:#0645AD;">BBa_K3889111</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p>yqcG | + | <p>yqcG toxin casette</p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889112">BBa_K3889112</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889112" target="_blank" style="color:#0645AD;">BBa_K3889112</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p>yqcF cassette</p> | + | <p>yqcF antitoxin cassette</p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889120">BBa_K3889120</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889120" target="_blank" style="color:#0645AD;">BBa_K3889120</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p> | + | <p>YtvA expression cassette</p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889121">BBa_K3889121</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889121" target="_blank" style="color:#0645AD;">BBa_K3889121</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p> | + | <p>bovine pancreatic DNase 1 toxin cassette</p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889122">BBa_K3889122</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889122" target="_blank" style="color:#0645AD;">BBa_K3889122</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889127">BBa_K3889127</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889127" target="_blank" style="color:#0645AD;">BBa_K3889127</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p> | + | <p>bpDNase1+mfLon_pdtB</p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889130">BBa_K3889130</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889130" target="_blank" style="color:#0645AD;">BBa_K3889130</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p>Spacer Cassette for Terminator check in Bacillus subtilis</p> | + | <p>Spacer Cassette for Terminator check in <em>Bacillus subtilis</em></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889131">BBa_K3889131</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889131" target="_blank" style="color:#0645AD;">BBa_K3889131</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p> | + | <p>BBa_B0010 terminator check cassette</p> |
</td> | </td> | ||
<td> | <td> | ||
Line 1,063: | Line 1,071: | ||
</td> | </td> | ||
<td> | <td> | ||
− | <p>Replacing spacer with terminator in <a href="http://parts.igem.org/Part:BBa_K3889130">BBa_K3889130</a> for checking terminator efficiency of <a href="http://parts.igem.org/Part:BBa_B0010" | + | <p>Replacing spacer with terminator in <a href="http://parts.igem.org/Part:BBa_K3889130" target="_blank" style="color:#0645AD;">BBa_K3889130</a> for checking terminator efficiency of <a href="http://parts.igem.org/Part:BBa_B0010" target="_blank" style="color:#0645AD;">BBa_B0010</a></p> |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889132">BBa_K3889132</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889132" target="_blank" style="color:#0645AD;">BBa_K3889132</a></p> |
</td> | </td> | ||
<td> | <td> | ||
− | <p> | + | <p>BBa_B0010-nagP fused terminator check cassette</p> |
</td> | </td> | ||
<td> | <td> | ||
Line 1,077: | Line 1,085: | ||
</td> | </td> | ||
<td> | <td> | ||
− | <p>Replacing spacer with terminator in <a href="http://parts.igem.org/Part:BBa_K3889130">BBa_K3889130</a> for checking terminator efficiency of <a href="http://parts.igem.org/Part:BBa_K3889070">BBa_K3889070</a></p> | + | <p>Replacing spacer with terminator in <a href="http://parts.igem.org/Part:BBa_K3889130" target="_blank" style="color:#0645AD;">BBa_K3889130</a> for checking terminator efficiency of <a href="http://parts.igem.org/Part:BBa_K3889070" target="_blank" style="color:#0645AD;">BBa_K3889070</a></p> |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889150">BBa_K3889150</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889150" target="_blank" style="color:#0645AD;">BBa_K3889150</a></p> |
</td> | </td> | ||
<td> | <td> | ||
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<tr> | <tr> | ||
<td> | <td> | ||
− | <p><a href="http://parts.igem.org/Part:BBa_K3889151">BBa_K3889151</a></p> | + | <p><a href="http://parts.igem.org/Part:BBa_K3889151" target="_blank" style="color:#0645AD;">BBa_K3889151</a></p> |
</td> | </td> | ||
<td> | <td> | ||
Line 1,111: | Line 1,119: | ||
</table> | </table> | ||
</div> | </div> | ||
− | < | + | <h2>Improvement:</h2> |
− | < | + | <h3>Introduction:</h3> |
− | + | ||
− | + | <p>While engineering any new circuit, there is always a need for <strong>well-characterized</strong> and <strong>predictable parts</strong>. Not only should the circuit function as expected, but it should also be orthogonal to irrelevant cell processes, thereby increasing the need to have efficient production and, in some cases, more importantly, efficient termination. While there are several well-studied and efficient terminators for <em>E. coli</em>, we found no specific efficient single terminator on the iGEM registry that could stand out for <em>B. subtilis</em> chassis. Hence, we decided to improvise a terminator which might fulfil this gap.</p> | |
− | + | ||
− | <p>While engineering any new circuit, there is always a need for well-characterized and predictable parts. Not only should the circuit function as expected, but it should also be orthogonal to irrelevant cell processes, thereby increasing the need to have efficient production and, in some cases, more importantly, efficient termination. While there are several well-studied and efficient terminators for <em>E.coli</em>, we found no specific efficient single terminator on the iGEM registry that could stand out for <em>B.subtilis</em> chassis. Hence, we decided to improvise a terminator which might fulfil this gap.</p> | + | |
− | < | + | <h3>Measuring efficiency:</h3> |
<p>The experiment is divided into two cassettes: one reference and the other is a test cassette containing a terminator whose efficiency needs to be determined as shown by Gale et al.[1].</p> | <p>The experiment is divided into two cassettes: one reference and the other is a test cassette containing a terminator whose efficiency needs to be determined as shown by Gale et al.[1].</p> | ||
<figure class="col-12 col-md-8 p-5 m-auto"> | <figure class="col-12 col-md-8 p-5 m-auto"> | ||
− | <img src="./ | + | <img src="https://static.igem.org/mediawiki/2021/b/b7/T--IISER-Tirupati_India--Improvement1.jpeg" alt="Spacer Cassette for Terminator check" style="width:100%"> |
+ | <figcaption class="text-center p-3">Fig 1. Spacer Cassette for Reference</figcaption> | ||
</figure> | </figure> | ||
− | <p>The reference and the test cassette provide us the expression levels of both the fluorescent proteins which could be compared to tell us how efficiently the terminator is working.</p> | + | |
+ | <figure class="col-12 col-md-8 p-5 m-auto"> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/a/a7/T--IISER-Tirupati_India--Improvement2.jpeg" alt="Spacer replaced by BBa_B0010 " style="width:100%"> | ||
+ | <figcaption class="text-center p-3">Fig 2. Spacer replaced by BBa_B0010</figcaption> | ||
+ | </figure> | ||
+ | <figure class="col-12 col-md-8 p-5 m-auto"> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/e/e7/T--IISER-Tirupati_India--Improvement3.jpeg" alt="Spacer replaced by BBa_K3889070" style="width:100%"> | ||
+ | <figcaption class="text-center p-3">Fig 3. Spacer replaced by BBa_K3889070</figcaption> | ||
+ | </figure> | ||
+ | <p>The <strong>device/reference</strong> (Fig 1.) and the test cassette (Fig 2 and 3.) provide us the expression levels of both the fluorescent proteins which could be compared to tell us how efficiently the terminator is working.</p> | ||
<p><br /><br /></p> | <p><br /><br /></p> | ||
− | <p>Formulae for terminator efficiency | + | <p>Formulae for terminator efficiency [1] </p> |
\begin{equation}\tag{1}TE_{Device}=\frac{mCherry_{0}}{sfGFP_{0}}\end{equation} | \begin{equation}\tag{1}TE_{Device}=\frac{mCherry_{0}}{sfGFP_{0}}\end{equation} | ||
Line 1,136: | Line 1,152: | ||
$mCherry_{0}\rightarrow$ mCherry produced by device without terminator<br><br> | $mCherry_{0}\rightarrow$ mCherry produced by device without terminator<br><br> | ||
$sfGFP_{0}\rightarrow$ sfGFP produced by device without terminator<br><br> | $sfGFP_{0}\rightarrow$ sfGFP produced by device without terminator<br><br> | ||
− | Using the device without any changes, $TE_{Device}$ can be calculated which gives the expression of <br>$mCherry$ in absence of a terminator.<br><br> | + | Using the <strong>device</strong> without any changes, $TE_{Device}$ can be calculated which gives the expression of <br>$mCherry$ in absence of a terminator.<br><br> |
− | \begin{equation}\tag{2} TE=100-\left[\left(\frac{mCherry}{sfGPF}\right)\times\left(\frac{1}{TE_{Device}}\right)\times100\right]\end{equation} | + | <div class="col-12 table-responsive" , style="font-size:20px;"> |
+ | \begin{equation}\tag{2} TE=100-\left[\left(\frac{mCherry}{sfGPF}\right)\times\left(\frac{1}{TE_{Device}}\right)\times100\right]\end{equation} </div> <br><br> | ||
where, <br><br> | where, <br><br> | ||
$mCherry$ $\rightarrow$ mCherry produced by device with the terminator that needs to checked<br><br> | $mCherry$ $\rightarrow$ mCherry produced by device with the terminator that needs to checked<br><br> | ||
$sfGFP$ $\rightarrow$ sfGFP produced by device with the terminator that needs to checked<br><br> | $sfGFP$ $\rightarrow$ sfGFP produced by device with the terminator that needs to checked<br><br> | ||
− | < | + | <h3>d-score:</h3> |
− | + | ||
<p>For <em>E. coli </em>terminators d'Aubenton Carafa [3] gave a scoring system as shown below:</p> | <p>For <em>E. coli </em>terminators d'Aubenton Carafa [3] gave a scoring system as shown below:</p> | ||
− | <p>$d=96.59 \times \frac{-\Delta G/(kcal/mol)}{ | + | <p>$d=96.59 \times \frac{-\Delta G/(kcal/mol)}{n_{SL}} + 18.16 \times T_{score} -116.87$</p> |
<p>Where </p> | <p>Where </p> | ||
<p>d is the d-score</p> | <p>d is the d-score</p> | ||
<p>$-\Delta G$ is the Gibbs free energy of stem-loop formation in kcal/mole</p> | <p>$-\Delta G$ is the Gibbs free energy of stem-loop formation in kcal/mole</p> | ||
− | <p> | + | <p>n<sub>SL</sub> is the length of the stem loop</p> |
− | <p> | + | <p>T<sub>Score</sub> is the score for T-stretch of the terminators</p> |
<p>Coefficients are according to fitting the d'Aubenton Carafa’s model </p> | <p>Coefficients are according to fitting the d'Aubenton Carafa’s model </p> | ||
Line 1,161: | Line 1,177: | ||
<p>$x_i = 0.6 \times x_{i-1}$ if $i^{th}$ nucleotide is not thymine</p> | <p>$x_i = 0.6 \times x_{i-1}$ if $i^{th}$ nucleotide is not thymine</p> | ||
− | <p> | + | <p>This scoring system was modified by de Hoon et al. [2] for <em>Bacillus subtilis</em> as per their model which is as follows:</p> |
− | <p>$d=7.90 \times \frac{-\Delta G/(kcal/mol)}{ | + | <p>$d=7.90 \times \frac{-\Delta G/(kcal/mol)}{n_{SL}} + 2.67 \times T_{score} -14.91$</p> |
<p>Where </p> | <p>Where </p> | ||
<p>d is the d-score</p> | <p>d is the d-score</p> | ||
<p>$-\Delta$ G is the Gibbs free energy of stem-loop formation in kcal/mole</p> | <p>$-\Delta$ G is the Gibbs free energy of stem-loop formation in kcal/mole</p> | ||
− | <p> | + | <p>n <sub>SL</sub> is the length of the stem loop</p> |
<p>T<sub>Score</sub> is the score for T-stretch of the terminators</p> | <p>T<sub>Score</sub> is the score for T-stretch of the terminators</p> | ||
<p>Coefficients are according to fitting the model </p> | <p>Coefficients are according to fitting the model </p> | ||
− | <p>Here the | + | <p>Here the T<sub>Score</sub> is calculated as follows:</p> |
<p>$T= \sum\limits_{i=0}^{\ 14} e^{- \lambda _i} \delta_i$</p> | <p>$T= \sum\limits_{i=0}^{\ 14} e^{- \lambda _i} \delta_i$</p> | ||
Line 1,179: | Line 1,195: | ||
<p>$\delta_i = 1$ if $i^{th}$ nucleotide is thymine</p> | <p>$\delta_i = 1$ if $i^{th}$ nucleotide is thymine</p> | ||
− | <p> | + | <p>As the <strong>d-score</strong> takes into account the <strong>Gibbs free energy</strong>,<strong> length of the stem-loop</strong> and the <strong>richness of thymine in the T-stretch</strong> which are essential for a rho independent terminator. Hence, the d-score can provide a rough idea about how good a terminator is. In other words, the <strong>higher the d-score higher will be the terminator efficiency</strong>.[3]</p> |
− | < | + | <h3>Improvement:</h3> |
− | + | <p>We decided to improve <a href="http://parts.igem.org/Part:BBa_B0010" target="_blank" style="color:#0645AD;">BBa_B0010</a> in order to make a strong terminator which can be used for primarily the <em>B. subtilis</em> chassis while still retaining its efficiency in <em>E. coli</em>. For doing this we modified the tail of <a href="http://parts.igem.org/Part:BBa_B0010" target="_blank" style="color:#0645AD;">BBa_B0010</a> and fused another rho-independent terminator from the <em>B. subtilis</em> genome on the basis of its d-Score. </p> | |
− | <p>We decided to improve BBa_B0010 in order to make a strong terminator which can be used for primarily the B.subtilis chassis while still retaining its efficiency in E.coli. For doing this we modified the tail of | + | |
− | <p>From a list of 425 native B.subtilis terminators taken from the study conducted by | + | <p>From a list of <strong>425 native B.subtilis terminators</strong> taken from the study conducted by de Hoon et al [2], we calculated the d-score of each terminator to get a rough idea of their efficiency which is in the <a href="https://static.igem.org/mediawiki/2021/d/d8/T--IISER-Tirupati_India--improvement.zip" style="color:#0645AD;"> Data file </a> containing both data as well as T-stretch calculator python file. Based on the results the <strong>highest d-score= 5.666126119 </strong>was of the terminator belonging to the gene <strong>nagP</strong>. Both <a href="http://parts.igem.org/Part:BBa_B0010" target="_blank" style="color:#0645AD;">BBa_B0010</a> and nagP terminators were ligated to form a double terminator.</p> |
− | <p>Based on our calculations, we decided to go with nagP terminator. We modified the end regions of BBa_B0010 and ligated to it the nagP terminator to create an improved version( | + | <p>Based on our calculations, we decided to go with nagP terminator. We modified the end regions of <a href="http://parts.igem.org/Part:BBa_B0010" target="_blank" style="color:#0645AD;">BBa_B0010</a> and ligated to it the nagP terminator to create an improved version(<a href="http://parts.igem.org/Part:BBa_K3889070" target="_blank" style="color:#0645AD;">BBa_K3889070</a>). Using the server <a href="http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi" target="_blank" style="color:#0645AD;">RNAFold</a> we calculated the minimum energy to show in silico that the improved terminator will have more negative Minimum Free energy as shown. </p> |
<table class="table table-bordered table-striped"> | <table class="table table-bordered table-striped"> | ||
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<p>The predicted structure for these two terminators as given by RnaFold server is:</p> | <p>The predicted structure for these two terminators as given by RnaFold server is:</p> | ||
<ol> | <ol> | ||
− | <li>BBa_B0010: | + | <li> BBa_B0010: |
<figure class="col-12 col-md-8 p-5 m-auto"> | <figure class="col-12 col-md-8 p-5 m-auto"> | ||
− | <img src="./ | + | <img src="https://static.igem.org/mediawiki/2021/e/e5/T--IISER-Tirupati_India--BBa_B0010.png" alt="BBa_B0010 from RnaFold" style="width:100%"> |
+ | <figcaption class="text-center p-3">Fig 4. BBa_B0010 from RnaFold</figcaption> | ||
</figure> | </figure> | ||
</li> | </li> | ||
− | <li>BBa_B0010+nagP: | + | <li> BBa_B0010+nagP: |
<figure class="col-12 col-md-8 p-5 m-auto"> | <figure class="col-12 col-md-8 p-5 m-auto"> | ||
− | <img src="./ | + | <img src="https://static.igem.org/mediawiki/2021/a/a8/T--IISER-Tirupati_India--BBa_B0010%2BnagP.png" alt="Fused BBa_B0010+nagP from RnaFold" style="width:100%"><span id="6"></span> |
+ | <figcaption class="text-center p-3">Fig 5. Fused BBa_B0010+nagP from RnaFold</figcaption> | ||
</figure> | </figure> | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | + | ||
− | < | + | <h3>References:</h3> |
− | < | + | <ol> |
− | < | + | <li> Gale, G. A. R., Wang, B., & McCormick, A. J. (2021). Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species. Frontiers in Microbiology, 11. https://doi.org/10.3389/fmicb.2020.624011 : </li> |
− | < | + | <li> de Hoon, M. J. L., Makita, Y., Nakai, K., & Miyano, S. (2005). Prediction of Transcriptional Terminators in Bacillus subtilis and Related Species. PLoS Computational Biology, 1(3), e25. https://doi.org/10.1371/journal.pcbi.0010025 </li> |
− | < | + | <li> Carafa, Y. d’Aubenton, Brody, E., & Thermes, C. (1990). Prediction of rho-independent Escherichia coli transcription terminators. Journal of Molecular Biology, 216(4), 835–858. https://doi.org/10.1016/s0022-2836(99)80005-9 </li> |
+ | </ol> | ||
</div> | </div> | ||
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<div class="quick_link_heading text-center mt-5 mb-5">Quick Links</div> | <div class="quick_link_heading text-center mt-5 mb-5">Quick Links</div> | ||
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<div class="d-flex flex-column"> | <div class="d-flex flex-column"> | ||
<a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Description">DESCRIPTION</a> | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Description">DESCRIPTION</a> | ||
− | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/ | + | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Blueprint">BLUEPRINT</a> |
<a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Engineering">ENGINEERING</a> | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Engineering">ENGINEERING</a> | ||
<a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Background">BACKGROUND</a> | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Background">BACKGROUND</a> | ||
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− | <h6 class="quick_link_heading">WETLAB</h6 | + | <h6 class="quick_link_heading">WETLAB</h6><br> |
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− | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/ | + | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Proof_Of_Concept">PROOF OF CONCEPT</a> |
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+ | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Excellence">EXCELLENCE</a> | ||
+ | |||
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<a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Education">EDUCATION</a> | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Education">EDUCATION</a> | ||
<a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Communication">COMMUNICATION</a> | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Communication">COMMUNICATION</a> | ||
− | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Inclusivity"> | + | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Inclusivity">INCLUSIVITY</a> |
<a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Sustainable">SUSTAINABLE DEVELOPMENT</a> | <a class="quick_link" href="https://2021.igem.org/Team:IISER-Tirupati_India/Sustainable">SUSTAINABLE DEVELOPMENT</a> | ||
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Latest revision as of 03:18, 17 December 2021
Overview
Due to the novel approaches taken while pursuing this project, we found ourselves using new parts that had not been given in the iGEM registry. In total, we developed 37 new basic parts (either entirely new or by modifying previously existing ones). We strongly believe that these two collections can be exploited to their full potential by the subsequent iGEM generations.
Furthermore, we have designed and used multiple combinations of basic parts as given in the composite part section.These composite parts can be used in multiple settings depending on the requirements of the projects.
The parts used in our project are as follows:
Previous Parts used:
BioBrick ID |
Name |
Length |
Description |
pVeg |
97 bp |
Constitutive natural promoter under Sig A transcription factor |
|
P43 |
56 bp |
Constitutive natural promoter under Sig A and SigB transcription factor |
|
pxylA |
82 bp |
Xylose dependent natural promoter (repressed by xylR) |
|
Protein degradation tag B |
87 bp |
mf-Lon specific Protein Degradation Tag B (medium-strong) |
|
RBS |
11 bp |
RBS for B. subtilis |
|
RBS |
11 bp |
RBS for B. subtilis |
|
RBS |
11 bp |
RBS for B. subtilis |
|
RBS |
11 bp |
RBS for B. subtilis |
|
mCherry |
714 bp |
Red fluorescent tag |
|
Terminator |
129 bp |
Double terminator consisting of BBa_B0010 and BBa_B0012 |
|
Terminator |
80 bp |
Relatively weaker Terminator as compared to BBa_B0015 |
|
TMini |
68 bp |
Very short terminator in yeast with good performance |
|
Basic Parts:
Part No: |
Name |
Length |
Description |
Azurite BFP (BFP.B3) |
717 bp |
Blue fluorescent tag |
|
modified_sfGFP |
720 bp |
Green fluorescent tag |
|
SP126 |
52 bp |
Promoter from synthetic library of Bacillus subtilis |
|
SP146 |
52 bp |
Promoter from synthetic library of Bacillus subtilis |
|
SP200 |
52 bp |
Promoter from synthetic library of Bacillus subtilis |
|
pgsiB |
300 bp |
Natural promoter of Bacillus subtilis under control of general stress SigB transcription factor |
|
pGAL1 Yeast Promoter |
531 bp |
Galactose inducible yeast promoter |
|
P22 c2 repressor |
648 bp |
DNA binding that represses gene expression |
|
Steroid responsive Transcription Factor (SRTF1) |
567 bp |
Transcription factor that can bind to specific DNA sequence to repress gene expression and is inhibited by progesterone |
|
Human Ovastacin protease phosphomimic_A |
594 bp |
Epitope region of the human protease ovastacin containing active site for ZP2 protein cleavage |
|
Human Ovastacin protease phosphomimic_B |
594 bp |
Human protease ovastacin with phospho mimic tyrosine |
|
Human Ovastacin protease |
594 bp |
Human protease ovastacin with phospho mimic tyrosine and serine |
|
yqcG toxin |
1593 bp |
Toxin part of type 2 toxin-antitoxin system of Bacillus subtilis (DNase) |
|
ytvA light sensor |
783 bp |
Blue light sensor that positively regulates SigB |
|
bovine pancreatic DNase 1 |
846 bp |
Highly potential and efficient endonuclease (Toxin) |
|
mf-Lon protease |
2361 bp |
Lon Protease from Mesoplasma florum bacteria |
|
Human Zona pellucida ZP2 protein partial |
1800 bp |
ZP2 protein that is cleaved by ovastacin |
|
SRTF 1 Binding Site |
20 bp |
SRTF1 binds to this DNA sequence to negatively regulate gene expression |
|
Bacillus subtilis Spacer Sequence |
40 bp |
Spacer sequence without any promoter, RBS or terminator activity |
|
Bacillus subtilis TAT signal peptide PhoD |
168 bp |
Signal Peptide for TAT secretion system in Bacillus subtilis |
|
Bacillus subtilis TAT signal peptide YwbN |
84 bp |
Signal Peptide for TAT secretion system in Bacillus subtilis |
|
P2A Peptide Linker PTV |
66 bp |
Self cleaving peptide sequence which separates two proteins during translation in same operon |
|
Bacillus subtilis oriented double terminator nagP |
122 bp |
Fused product of nagP and BBa_B0010 (Improvement in BBa_B0010) |
|
P22 binding site A |
18 bp |
P22 binds to this DNA sequence to negatively regulate gene expression |
|
P22 binding site B |
18 bp |
P22 binds to this DNA sequence to negatively regulate gene expression |
|
P22 binding site C |
18 bp |
P22 binds to this DNA sequence to negatively regulate gene expression |
|
P22 binding site D |
18 bp |
P22 binds to this DNA sequence to negatively regulate gene expression |
|
P22 binding site E |
18 bp |
P22 binds to this DNA sequence to negatively regulate gene expression |
|
P22 binding site F |
18 bp |
P22 binds to this DNA sequence to negatively regulate gene expression |
|
P22 binding site G |
18 bp |
P22 binds to this DNA sequence to negatively regulate gene expression |
|
P22 binding site H |
18 bp |
P22 binds to this DNA sequence to negatively regulate gene expression |
|
P22 binding site I |
18 bp |
P22 binds to this DNA sequence to negatively regulate gene expression |
|
P22 binding site J |
18 bp |
P22 binds to this DNA sequence to negatively regulate gene expression |
|
P22 binding site K |
18 bp |
P22 binds to this DNA sequence to negatively regulate gene expression |
|
P22 binding site L |
18 bp |
P22 binds to this DNA sequence to negatively regulate gene expression |
|
XylR |
1050 bp |
pXylA repressor which provides xylose inducible gene expression |
|
yqcF |
576 bp |
Anti-toxin of yqcG (a kind of DNase) |
Composite Parts:
Part No: |
Name |
Length |
Description |
SRTF1 expression cassette |
1539 bp |
Constitutive production of SRTF1 being reported by mCherry |
|
P22 expression cassette |
1643 bp |
Progesterone inducible production due to fusion of SRTF1 binding site to the promoter reported by Azurite |
|
Ovastacin expression cassette |
1042 bp |
P22 controlled (indirect progesterone control) ovastacin production reported by sfGFP |
|
XylR Repressor Cassette |
2032 bp |
Constitutive production of XylR being reported by sfGFP |
|
yqcG toxin casette |
1815 bp |
Xylose inducible production of YqcG |
|
yqcF antitoxin cassette |
1548 bp |
Constitutive production of YqcF being reported by mCherry |
|
YtvA expression cassette |
1759 bp |
Constitutive production of YtvA being reported by mCherry |
|
bovine pancreatic DNase 1 toxin cassette |
1275 bp |
Light inducible production of bpDNase 1 |
|
mf-Lon cassette |
3339 bp |
Constitutive production of mf-Lon being reported by sfGFP |
|
bpDNase1+mfLon_pdtB |
933 bp |
bpDnase 1 fused with protein degradation tag of mf-Lon |
|
Spacer Cassette for Terminator check in Bacillus subtilis |
1722 bp |
Spacer in between of two reporters mCherry and sfGFP which provides with basal level expression of downstream genes in absence of a terminator |
|
BBa_B0010 terminator check cassette |
1762 bp |
Replacing spacer with terminator in BBa_K3889130 for checking terminator efficiency of BBa_B0010 |
|
BBa_B0010-nagP fused terminator check cassette |
1804 bp |
Replacing spacer with terminator in BBa_K3889130 for checking terminator efficiency of BBa_K3889070 |
|
SP200+SRTF1 Binding Site+ RBS |
83 bp |
SRTF1 binding site fused with SP200 promoter for progesterone inducible gene expression |
|
P43+P22 Binding Site L +RBS |
85 bp |
P22 binding site fused with P43 for P22 controlled gene expression |
Improvement:
Introduction:
While engineering any new circuit, there is always a need for well-characterized and predictable parts. Not only should the circuit function as expected, but it should also be orthogonal to irrelevant cell processes, thereby increasing the need to have efficient production and, in some cases, more importantly, efficient termination. While there are several well-studied and efficient terminators for E. coli, we found no specific efficient single terminator on the iGEM registry that could stand out for B. subtilis chassis. Hence, we decided to improvise a terminator which might fulfil this gap.
Measuring efficiency:
The experiment is divided into two cassettes: one reference and the other is a test cassette containing a terminator whose efficiency needs to be determined as shown by Gale et al.[1].
![Spacer Cassette for Terminator check](https://static.igem.org/mediawiki/2021/b/b7/T--IISER-Tirupati_India--Improvement1.jpeg)
![Spacer replaced by BBa_B0010](https://static.igem.org/mediawiki/2021/a/a7/T--IISER-Tirupati_India--Improvement2.jpeg)
![Spacer replaced by BBa_K3889070](https://static.igem.org/mediawiki/2021/e/e7/T--IISER-Tirupati_India--Improvement3.jpeg)
The device/reference (Fig 1.) and the test cassette (Fig 2 and 3.) provide us the expression levels of both the fluorescent proteins which could be compared to tell us how efficiently the terminator is working.
Formulae for terminator efficiency [1]
\begin{equation}\tag{1}TE_{Device}=\frac{mCherry_{0}}{sfGFP_{0}}\end{equation}where,
$mCherry_{0}\rightarrow$ mCherry produced by device without terminator
$sfGFP_{0}\rightarrow$ sfGFP produced by device without terminator
Using the device without any changes, $TE_{Device}$ can be calculated which gives the expression of
$mCherry$ in absence of a terminator.
where,
$mCherry$ $\rightarrow$ mCherry produced by device with the terminator that needs to checked
$sfGFP$ $\rightarrow$ sfGFP produced by device with the terminator that needs to checked
d-score:
For E. coli terminators d'Aubenton Carafa [3] gave a scoring system as shown below:
$d=96.59 \times \frac{-\Delta G/(kcal/mol)}{n_{SL}} + 18.16 \times T_{score} -116.87$
Where
d is the d-score
$-\Delta G$ is the Gibbs free energy of stem-loop formation in kcal/mole
nSL is the length of the stem loop
TScore is the score for T-stretch of the terminators
Coefficients are according to fitting the d'Aubenton Carafa’s model
The TScore is calculated as follows:
$T_{score}= \sum\limits_{i=0}^{\ 14} x _i$
Where
$x_0 = 0.9$
$x_i = 0.9$ if $i^{th}$ nucleotide is thymine
$x_i = 0.6 \times x_{i-1}$ if $i^{th}$ nucleotide is not thymine
This scoring system was modified by de Hoon et al. [2] for Bacillus subtilis as per their model which is as follows:
$d=7.90 \times \frac{-\Delta G/(kcal/mol)}{n_{SL}} + 2.67 \times T_{score} -14.91$
Where
d is the d-score
$-\Delta$ G is the Gibbs free energy of stem-loop formation in kcal/mole
n SL is the length of the stem loop
TScore is the score for T-stretch of the terminators
Coefficients are according to fitting the model
Here the TScore is calculated as follows:
$T= \sum\limits_{i=0}^{\ 14} e^{- \lambda _i} \delta_i$
Where
$\lambda _i = 0.144$ as per the fitting of the model
$\delta_i = 0$ if $i^{th}$ nucleotide is not thymine
$\delta_i = 1$ if $i^{th}$ nucleotide is thymine
As the d-score takes into account the Gibbs free energy, length of the stem-loop and the richness of thymine in the T-stretch which are essential for a rho independent terminator. Hence, the d-score can provide a rough idea about how good a terminator is. In other words, the higher the d-score higher will be the terminator efficiency.[3]
Improvement:
We decided to improve BBa_B0010 in order to make a strong terminator which can be used for primarily the B. subtilis chassis while still retaining its efficiency in E. coli. For doing this we modified the tail of BBa_B0010 and fused another rho-independent terminator from the B. subtilis genome on the basis of its d-Score.
From a list of 425 native B.subtilis terminators taken from the study conducted by de Hoon et al [2], we calculated the d-score of each terminator to get a rough idea of their efficiency which is in the Data file containing both data as well as T-stretch calculator python file. Based on the results the highest d-score= 5.666126119 was of the terminator belonging to the gene nagP. Both BBa_B0010 and nagP terminators were ligated to form a double terminator.
Based on our calculations, we decided to go with nagP terminator. We modified the end regions of BBa_B0010 and ligated to it the nagP terminator to create an improved version(BBa_K3889070). Using the server RNAFold we calculated the minimum energy to show in silico that the improved terminator will have more negative Minimum Free energy as shown.
BBa_B0010 |
BBa_B0010+nagP |
|
Minimum Free Energy (kcal/mol) |
-40 |
-64.6 |
The predicted structure for these two terminators as given by RnaFold server is:
- BBa_B0010:
Fig 4. BBa_B0010 from RnaFold - BBa_B0010+nagP:
Fig 5. Fused BBa_B0010+nagP from RnaFold
References:
- Gale, G. A. R., Wang, B., & McCormick, A. J. (2021). Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species. Frontiers in Microbiology, 11. https://doi.org/10.3389/fmicb.2020.624011 :
- de Hoon, M. J. L., Makita, Y., Nakai, K., & Miyano, S. (2005). Prediction of Transcriptional Terminators in Bacillus subtilis and Related Species. PLoS Computational Biology, 1(3), e25. https://doi.org/10.1371/journal.pcbi.0010025
- Carafa, Y. d’Aubenton, Brody, E., & Thermes, C. (1990). Prediction of rho-independent Escherichia coli transcription terminators. Journal of Molecular Biology, 216(4), 835–858. https://doi.org/10.1016/s0022-2836(99)80005-9
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