Difference between revisions of "Team:TUDelft/Notebook"

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  <body>
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    <div class="Banner container-fluid text-center mb-0 align-items-center ">
 +
 
 +
      <div class="display-2 mb-0">  
 +
        <br>
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        <br>
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        <br>
 +
        <br>
 +
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      </div>     
 +
    </div>   
 +
    </div>
 +
  <div class="centerjustify">
 +
    <h2>Orthogonal Replication</h2>
 +
    <br>
 +
    <table id="tablenotebook" class="notebook">
 +
      <tr id="category">
 +
        <th style="background-color:transparent; color:black;"></th>
 +
        <th style="background-color:transparent; color:black;" colspan=2> Cloning</th>
 +
        <th style="background-color:transparent; color:black;" colspan=2>Testing of Plasmid</th>
 +
      </tr>
 +
      <tr>
 +
        <td class="month">April</td>
 +
        <td rowspan=4 class="modalTD">
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal1">Gibson Assembly of Gene of Interest Plasmid</button>
 +
 
 +
          <!-- The Modal -->
 +
          <div id="myModal1" class="modal">
 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
 +
                <span class="close">×</span>
 +
                <h2>Gibson Assembly of Gene of Interest Plasmid</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>We wanted to construct a Gene of Interest Plasmid. The Gene of Interest of Plasmid should have OriL, gene of interest, selection marker and OriR. The gene of interest was GFP in our case. We had a plasmid with OriL-GFP-OriR and therefore we designed primers to clone Kanamycin in. The Gene of Interest Plasmid is the plasmid that will be replicated with the phi29 machinery. </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td> <a href="https://2019.igem.org/Team:TUDelft/Experiments">Nanodrop DNA quantification</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">Plasmid Isolation</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Phusion PCR</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Restriction Digestion</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Heat Shock Transformation</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">KOD Extreme PCR</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Colony Polymerase PCR (GoTaq)</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Glycerol Stock</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Gibson Assembly</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">DNA Gel Purification</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">DNA Gel Electrophoresis</a>
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>Our plasmid p01 contained GFP flanked by OriL and OriR. We wanted to clone a kanamycin gene from plasmid p03 inside this to use as a selection marker. After Gibson Assembly, the plasmid was transformed into <i>E. coli</i> top 10 cells and plated on LB + Kan.</td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>There were no plates with colonies, therefore Gibson Assembly has not worked as no cells expressed the Kanamycin gene. </td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
 
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
        </td>
 +
        <td rowspan=4 class="modalTD" >
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal2">Gibson Assembly of Support Plasmid</button>
 +
 
 +
          <!-- The Modal -->
 +
          <div id="myModal2" class="modal">
 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
 +
                <span class="close">×</span>
 +
                <h2>Gibson Assembly of Support Plasmid</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>We wanted to construct the Support Plasmid. We have two plasmids each containing two of the four replication machinery. We wanted to put them on one plasmid to minimize the amount of plasmids to be transformed into our construct.</td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td> <a href="https://2019.igem.org/Team:TUDelft/Experiments">Nanodrop DNA quantification</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">Plasmid Isolation</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">Phusion PCR</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">Restriction Digestion</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Heat Shock Transformation</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">KOD Extreme PCR</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">Colony Polymerase PCR (GoTaq)</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Glycerol Stock</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Gibson Assembly</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">DNA Gel Purification</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">DNA Gel Electrophoresis</a>
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>We wanted to assembly the replication machinery on one plasmid through Gibson Assembly. We possess one plasmid (p04) with p5 and p6 and one plasmid with DNAP and TP (p02) and therefore they were cloned together. After Gibson Assembly, the plasmid was transformed into <i>E. coli</i> top 10 cells and plated on LB + Amp.</td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>There were no plates with colonies, therefore Gibson Assembly has not worked as no cells expressed the Ampicilin gene. </td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
        </td>
 +
        <td colspan=2></td>
 +
      </tr>
 +
      <tr>
 +
        <td class="month">May</td>
 +
 
 +
 
 +
        <td colspan=2></td>
 +
      </tr>
 +
      <tr>
 +
        <td class="month">June</td>
 +
        <td colspan=2></td>
 +
 
 +
      </tr>
 +
      <tr>
 +
        <td class="month">July</td>
 +
 
 +
 
 +
 
 +
        <td colspan=2 class="modalTD">
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal3">Brute Force Experiments</button>
 +
 
 +
          <!-- The Modal -->
 +
          <div id="myModal3" class="modal">
 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
 +
                <span class="close">×</span>
 +
                <h2>Brute Force Experiments</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>Testing orthogonal replication in vivo</td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td> <a href="https://2019.igem.org/Team:TUDelft/Experiments">Transformation</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">IPTG stock</a>
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>Although the construction of the plasmids using Gibson Assembly did not succeed, we transformed a combination of constructs to determine whether in vivo replication would occur. We induced it with no IPTG, 10 µM IPTG and 50 µM IPTG to enhance protein expression. We transformed in the following constructs into BL21(DE3) Rosetta 2 cells:
 +
                      <ul>
 +
                        <li>p02 (DNAP + TP) + p04 (p5 +p6) + oriL-GFP-oriR</li>
 +
                        <li>p02 (DNAP - TP) + oriL-GFP-oriR</li>
 +
                        <li>oriL-GFP-oriR</li>
 +
                        <li>p01 (GFP) + p02 (DNAP + TP) + p04 (p5 + p6)</li>
 +
                      </ul></td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>There were no colonies on most of the plates. Inconclusive result.</td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
 
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
        </td>
 +
      </tr>
 +
      <tr>
 +
        <td class="month">August</td>
 +
        <td rowspan=2 colspan=2 class="modalTD">
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal4">MoClo Assembly</button>
 +
 
 +
          <!-- The Modal -->
 +
          <div id="myModal4" class="modal">
 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
 +
                <span class="close">×</span>
 +
                <h2>MoClo Assembly</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>Assembling the parts of the replication machinery separately with MoClo</td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td> <a href="https://2019.igem.org/Team:TUDelft/Experiments">Nanodrop DNA quantification</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">Plasmid Isolation</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">MoClo</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Heat Shock Transformation</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">KOD Extreme PCR</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">Colony Polymerase PCR (GoTaq)</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Glycerol Stock</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">DNA Gel Electrophoresis</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Sequencing</a>
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>First all the level 0's were constructed for the orthogonal replication module.
 +
                       
 +
                        Subsequently, the level 0's were used to assembly the following level 1 constructs:
 +
                      <ul>
 +
                        <li><a href="https://2019.igem.org/Team:TUDelft/Experiments">IPTG stock</a> 0.1 T7 promoter - Uni RBS - DNAP - WT T7 terminator</li>
 +
                        <li>0.5 T7 promoter - Uni RBS - DNAP - WT T7 terminator</li>
 +
                        <li>WT T7 promoter - Uni RBS - DNAP - WT T7 terminator</li>
 +
                        <li>0.1 T7 promoter - Uni RBS - TP - WT T7 terminator</li>
 +
                        <li>0.5 T7 promoter - Uni RBS - TP - WT T7 terminator</li>
 +
                        <li>WT T7 promoter - Uni RBS - TP - WT T7 terminator</li>
 +
                        <li>0.1 T7 promoter - Uni RBS - p5 - WT T7 terminator</li>
 +
                        <li>0.5 T7 promoter - Uni RBS - p5 - WT T7 terminator</li>
 +
                        <li>WT T7 promoter - Uni RBS - p5 - WT T7 terminator</li>
 +
                        <li>0.1 T7 promoter - Uni RBS - p6 - WT T7 terminator</li>
 +
                        <li>0.5 T7 promoter - Uni RBS - p6 - WT T7 terminator</li>
 +
                        <li>WT T7 promoter - Uni RBS - p6 - WT T7 terminator</li>
 +
                        <li>0.1 T7 promoter - Uni RBS - GFP - WT T7 terminator</li>
 +
                        <li>0.5 T7 promoter - Uni RBS - GFP - WT T7 terminator</li>
 +
                        <li>WT T7 promoter - Uni RBS - GFP - WT T7 terminator</li>
 +
                      </ul>
 +
 
 +
                    </td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>All composite parts are confirmed by sequencing</td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
 
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
        </td>
 +
        <td colspan=2 class="modalTD">
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal5">Toxicity Experiments 1</button>
 +
 
 +
          <!-- The Modal -->
 +
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 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
 +
                <span class="close">×</span>
 +
                <h2>Toxicity Experiments 1</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>Determinig whether the orthogonal replication constructs could be toxic to the cell</td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td>
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Heat Shock Transformation</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">OD600 Measurement</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">IPTG stock</a>
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>The following plasmids were transformed into BL21(DE3) Rosetta 2 separately:
 +
                      <ul>
 +
                        <li>p01 (GFP as control)</li>
 +
                        <li>p02 (DNAP + TP)</li>
 +
                        <li>p03 (Kan)</li>
 +
                        <li>p04 (p5 + p6)</li>
 +
                      </ul>
 +
                      <br>
 +
                      Transformation was carried out to determine whether the failure of the Gibson Assembly constructions were caused by toxicity of the orthogonal replication proteins.
 +
 
 +
                    </td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>The OD measurements of p02 and p04 dropped very quickly after 3 hours. However, the results are inconclusive due to lack of selection </td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
 
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
        </td>
 +
 
 +
      </tr>
 +
      <tr>
 +
        <td rowspan=2 class="month">September</td>
 +
 
 +
 
 +
        <td rowspan=2 class="modalTD">
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal6">Toxicity Experiments 2</button>
 +
 
 +
          <!-- The Modal -->
 +
          <div id="myModal6" class="modal">
 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
 +
                <span class="close">×</span>
 +
                <h2>Toxicity Experiments 2</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>Determinig whether the orthogonal replication constructs could be toxic to the cell</td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td>
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Heat Shock Transformation</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">OD600 Measurement</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">IPTG stock</a>
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>A different method and type of cell were used to determine how this would influence the suspected toxicity. The cells used in the experiment were BL21 (DE3) pLysS which have less leaky expression of T7 RNAP than BL21 (DE3) and therefore are suspected to survive longer. To determine whether this type of cell works better, we transformed the plasmids separately in BL21 (DE3) pLysS and measured the OD600 overnight to determine the viability of the cells containing the following plasmid:
 +
                      <ul>
 +
                        <li>p01 (GFP as control)</li>
 +
                        <li>p02 (DNAP + TP)</li>
 +
                        <li>p03 (Kan)</li>
 +
                        <li>p04 (p5 + p6)</li>
 +
 
 +
                      </ul>
 +
 
 +
                    </td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>The cells containing the plasmids did not grow overnight</td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
 
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
        </td>
 +
        <td rowspan =2 class="modalTD">
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal7">Characterization of Level 1</button>
 +
 
 +
          <!-- The Modal -->
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 +
 
 +
            <!-- Modal content -->
 +
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              <div class="modal-header">
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                <span class="close">×</span>
 +
                <h2>Characterization of Level 1</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>Characterize the different promoters strengths in the assembled constructs</td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td>
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Expressing phi29 proteins in PUREfrex system</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Purifying phi29 proteins for SDS PAGE</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Mass Spectrometry Preparation</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">SDS Page</a>
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>The following constructs were expressed in vitro and run on a protein gel to determine whether there was amplification of the gene:
 +
                      <ul>
 +
                        <li>0.1 T7 promoter - U RBS - DNAP - WT T7 terminator</li>
 +
                        <li>0.5 T7 promoter - U RBS - DNAP - WT T7 terminator</li>
 +
                        <li>WT T7 promoter - U RBS - DNAP - WT T7 terminator</li>
 +
                        <li>0.1 T7 promoter - U RBS - TP - WT T7 terminator</li>
 +
                        <li>0.5 T7 promoter - U RBS - TP - WT T7 terminator</li>
 +
                        <li>WT T7 promoter - U RBS - TP - WT T7 terminator</li>
 +
                        <li>0.1 T7 promoter - U RBS - p5 - WT T7 terminator</li>
 +
                        <li>0.5 T7 promoter - U RBS - p5 - WT T7 terminator</li>
 +
                        <li>WT T7 promoter - U RBS - p5 - WT T7 terminator</li>
 +
                        <li>0.1 T7 promoter - U RBS - p6 - WT T7 terminator</li>
 +
                        <li>0.5 T7 promoter - U RBS - p6 - WT T7 terminator</li>
 +
                        <li>WT T7 promoter - U RBS - p6 - WT T7 terminator</li></ul>
 +
                      <br>
 +
                      Subsequently, a fraction of the sample was analyzed with Mass Spectrometry.
 +
                    </td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>The strength of the promoter correlates with the amount of proteins transcribed. Read more <a href="https://2019.igem.org/Team:TUDelft/Results">here</a>.</td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
 
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
        </td>
 +
      </tr>
 +
 
 +
      <tr>
 +
 
 +
        <td colspan=2></td>
 +
 
 +
      </tr>
 +
 
 +
      <tr>
 +
        <td class="month">October</td>
 +
        <td> </td>
 +
        <td> </td>
 +
       
 +
        <td rowspan =2 class="modalTD">
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal9">Orthogonal Replication</button>
 +
 
 +
          <!-- The Modal -->
 +
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 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
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 +
                <h2>Orthogonal Replication</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>To replicate our constructs (OriL-GFP-Kan-OriR or OriL-GFP-OriR) in vitro</td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td>
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">DNA gel electrophoresis</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Coupled IVTT and DNA replication</a>
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>
 +
                      Our construct (OriL-GFP-Kan-OriR) combined our MoClo DNAP and TP plasmid were added with purifed p5 and p6 to determine whether replication would occur. In parallel, we added a plasmid containing DNAP and TP, in a mix containing a shorter construct: OriL-GFP-OriR together with pruified p5 and p6.
 +
                    </td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>Replication in vitro occurs! Read more <a href="https://2019.igem.org/Team:TUDelft/Results">here</a>.</td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
 
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
        </td>
 +
        <td rowspan=2>
 +
        </td>
 +
      </tr>
 +
    </table>
 +
    <br><br>
 +
    <h2>Controllable Expression</h2>
 +
    <br>
 +
    <table id="tablenotebook" class="notebook">
 +
      <tr id="category">
 +
        <th style="background-color:transparent; color:black;"></th>
 +
        <th style="background-color:transparent; color:black;">Cloning</th>
 +
        <th style="background-color:transparent; color:black;" colspan="3">Testing of Plasmid</th>
 +
      </tr>
 +
      <tr>
 +
        <td class="month">April</td>
 +
        <td></td>
 +
        <td colspan="3"></td>
 +
 
 +
      </tr>
 +
      <tr>
 +
        <td class="month">May</td>
 +
        <td></td>
 +
        <td colspan="3"></td>
 +
 
 +
      </tr>
 +
      <tr>
 +
        <td class="month">June</td>
 +
        <td class="modalTD" >
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal10">UBER Plasmid</button>
 +
 
 +
          <!-- The Modal -->
 +
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 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
 +
                <span class="close">×</span>
 +
                <h2>UBER Plasmid</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>Construction of the UBER Plasmid</td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td> <a href="https://2019.igem.org/Team:TUDelft/Experiments">Nanodrop DNA quantification</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">Plasmid Isolation</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">Phusion PCR</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">Restriction Digestion</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Heat Shock Transformation</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">Ligation</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Colony Polymerase PCR (GoTaq)</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Glycerol Stock</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">DNA Gel Purification</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">DNA Gel Electrophoresis</a>
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>We wanted to insert GFP in the UBER plasmid by restriction digestion and ligation. </td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>Check PCR did not result in the expected size on the gel</td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
        </td>
 +
        <td colspan="3"></td>
 +
 
 +
      </tr>
 +
      <tr>
 +
        <td class="month">July</td>
 +
 
 +
        <td></td>
 +
        <td colspan="3"></td>
 +
 
 +
      </tr>
 +
      <tr>
 +
        <td class="month">August</td>
 +
        <td rowspan=3 class="modalTD">
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal11">MoClo Assembly</button>
 +
 
 +
          <!-- The Modal -->
 +
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 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
 +
                <span class="close">×</span>
 +
                <h2>MoClo Assembly</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>Assembly all the different parts with MoClo</td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td> <a href="https://2019.igem.org/Team:TUDelft/Experiments">Nanodrop DNA quantification</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">Plasmid Isolation</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments">MoClo</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Heat Shock Transformation</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Colony Polymerase PCR (GoTaq)</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Glycerol Stock</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">DNA Gel Electrophoresis</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Sequencing</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Xgal Stock</a>
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>For Predictable & Transferable Expression, constructs were assembled by golden gate assembly based modular cloning systesm. First, individual parts were cloned in respective level 0 backbones.
 +
                      <ul>
 +
                        <li> T7 promoter </li>
 +
                        <li> 0.5 T7 promoter</li>
 +
                        <li> 0.1 T7 promoter </li>
 +
                        <li> P <sub>BHR</sub> promoter </li>
 +
                        <li> T7sp1 promoter </li>
 +
                        <li> 0.5 T7sp1 promoter </li>
 +
                        <li> 0.1 T7sp1 promoter </li>
 +
                        <li> P<sub>BHR</sub>sp1 promoter </li>
 +
                        <li> SarJ </li>
 +
                        <li> RiboJ </li>
 +
                        <li> Universal RBS </li>
 +
                        <li> TALEsp1 </li>
 +
                        <li> Harmonized GFP</li>
 +
                        <li> T7 terminator variant </li>
 +
                      </ul>
 +
                      <br>
 +
                      The level 0 parts were assembled into transcriptional units level 1 MoClo backbones:
 +
                      <br>
 +
                      <ul>
 +
                        <li>WT T7 promoter - Uni RBS - Harmonized GFP - WT T7 terminator</li>
 +
                        <li>L1_A: Promoter<sub>BHR</sub>sp1-RiboJ - Uni RBS - Juniper GFP - WT T7 terminator</li>
 +
                        <li>L1_B: T7_sp1 promoter - RiboJ - Uni RBS - Harmonized GFP - WT T7 terminator</li>
 +
                        <li>L1_C: Promoter<sub>BHR</sub> - Sarj-Uni RBS - TALEsp1 - WT T7 terminator variant</li>
 +
                        <li>L1_D: T7 promoter - SarJ-Uni RBS - TALEsp1 - WT T7 terminator variant </li>
 +
                        <li>L1_E: T7 promoter - RiboJ-Uni RBS - Harmonized GFP - WT T7 terminator</li>
 +
                        <li>L1_F: 0.5 T7 promoter - SarJ-Uni RBS- TALEsp1 - WT T7 terminator variant</li>
 +
                        <li>L1_G: 0.1 T7 promoter - SarJ-Uni RBS - TALEsp1- WT T7 terminator</li>
 +
                        <li>L1_H: 0.5 T7 promoter - RiboJ-Uni RBS - Harmonized GFP - WT T7 termniator</li>
 +
                      </ul>
 +
                      <br>
 +
                      The following multiple transcriptional units were assembled in MoClo level M backbones:
 +
                      <ul>
 +
                        <li>L1_C+L1_A </li>
 +
                        <li>L1_D+L1_B </li>
 +
                        <li>L1_D+L1_E </li>
 +
                        <li>L1_F+L1_H </li>
 +
                        <li>L1_G+L1_B </li>
 +
 
 +
                      </ul>
 +
 
 +
                    </td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>Read more <a href="https://2019.igem.org/Team:TUDelft/Results">here</a>.</td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
 
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
        <td class="modalTD">
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal14">Codon Harmonization</button>
 +
 
 +
          <!-- The Modal -->
 +
          <div id="myModal14" class="modal">
 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
 +
                <span class="close">×</span>
 +
                <h2>Codon Harmonization</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>Demonstration that the harmonized coding sequence generated from our modelling still 
 +
 
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td> <a href="https://2019.igem.org/Team:TUDelft/Experiments">Nanodrop DNA quantification</a>
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Plasmid Isolation</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Heat Shock Transformation</a>
 +
 
 +
 
 +
 
 +
 
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>
 +
                      The harmonized GFP construct (WT T7 promoter - Uni RBS - Harmonized GFP - WT T7 terminator) was transformed into BL21 pLysS cells together with a plasmid with regular GFP (positive control). As a negative control a cell without plasmid was used.
 +
                    </td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>The harmonized GFP works. Read more <a href="https://2019.igem.org/Team:TUDelft/Results">here</a>.</td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
 
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
 
 +
 
 +
        </td>
 +
        <td colspan="2"></td>
 +
 
 +
      </tr>
 +
      <tr>
 +
        <td class="month">September</td>
 +
 
 +
        <td rowspan=2 class="modalTD">
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal12">Copy Number Independence</button>
 +
 
 +
          <!-- The Modal -->
 +
          <div id="myModal12" class="modal">
 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
 +
                <span class="close">×</span>
 +
                <h2>Copy Number Independence</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>To demonstrate stable GFP expression despite change in copy number
 +
 
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td> <a href="https://2019.igem.org/Team:TUDelft/Experiments">Nanodrop DNA quantification</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Plasmid Isolation</a>,
 +
 
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">MoClo</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Heat Shock Transformation</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Colony Polymerase PCR (GoTaq)</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Glycerol Stock</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">DNA Gel Electrophoresis</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Sequencing</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Xgal Stock</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">OD600 Measurement</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">FACS</a>
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>Level M constructs (L1D + L1E, L1G + L1B) were both cloned in low and medium copy backbones. The constructs were then co-transformed with <a href="https://www.addgene.org/71428/">MK1283</a> which contains a plasmid harbouring a T7 RNAP mixed forward loop. Fluorescence was detected by FACS.
 +
                      <ul>
 +
                        <li>L1_A: Promoter<sub>BHR</sub>sp1- RiboJ- Uni RBS - Juniper GFP- T7 terminator</li>
 +
                        <li>L1_B: T7_sp1 promoter - RiboJ-UniRBS - Harmonized GFP - T7 terminator</li>
 +
                        <li>L1_C: Promoter<sub>BHR</sub> - Sarj-UniRBS - TALEsp1 - T7 terminator variance</li>
 +
                        <li>L1_D: T7 promoter - SarJ-UniRBS- TALEsp1 - T7 terminator variance </li>
 +
                        <li>L1_E: T7 promoter - RiboJ-UniRBS - Harmonized GFP -T7 terminator</li>
 +
                        <li>L1_F: 0.5 T7 promoter - SarJ-UniRBS- TALEsp1 - T7 terminator variance</li>
 +
                        <li>L1_G: 0.1 T7 promoter - SarJ-UniRBS - TALEsp1- T7 terminator</li>
 +
                        <li>L1_H: 0.5 T7 promoter - RiboJ-UniRBS- Harmonized GFP - T7 terminator</li>
 +
                      </ul>
 +
                    </td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>Read more <a href="https://2019.igem.org/Team:TUDelft/Results">here</a>.</td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
 
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
        </td>
 +
        <td rowspan=2 class="modalTD">
 +
          <!-- Trigger/Open The Modal -->
 +
          <button class="block modal-button" href="#myModal13">Transcriptional Variantions</button>
 +
 
 +
          <!-- The Modal -->
 +
          <div id="myModal13" class="modal">
 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
 +
                <span class="close">×</span>
 +
                <h2>Transcriptional Variantions</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>
 +
                      To demonstrate stable GFP expression despite change in transcriptional rates
 +
 
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td><a href="https://2019.igem.org/Team:TUDelft/Experiments">Nanodrop DNA quantification</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Plasmid Isolation</a>,
 +
 
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">MoClo</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Heat Shock Transformation</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Colony Polymerase PCR (GoTaq)</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Glycerol Stock</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">DNA Gel Electrophoresis</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Sequencing</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Xgal Stock</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">OD600 Measurement</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">FACS</a>
 +
 
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>
 +
                      Level M constructs (L1_C+L1_A, L1_D+L1_B, L1_D+L1_E, L1_F+L1_H, L1_G+L1_B) were characterized using FACS to demonstrate stable GFP expression when the ratio of transcriptional rates between GFP and TALE remained the same.
 +
 
 +
                      Here a quick reminder of what the constructs were:
 +
                      <ul>
 +
                        <li>L1_A: Promoter<sub>BHR</sub>sp1- RiboJ- Uni RBS - Juniper GFP- T7 terminator</li>
 +
                        <li>L1_B: T7_sp1 promoter - RiboJ-UniRBS - Harmonized GFP - T7 terminator</li>
 +
                        <li>L1_C: Promoter<sub>BHR</sub> - Sarj-UniRBS - TALEsp1 - T7 terminator variance</li>
 +
                        <li>L1_D: T7 promoter - SarJ-UniRBS- TALEsp1 - T7 terminator variance </li>
 +
                        <li>L1_E: T7 promoter - RiboJ-UniRBS - Harmonized GFP -T7 terminator</li>
 +
                        <li>L1_F: 0.5 T7 promoter - SarJ-UniRBS- TALEsp1 - T7 terminator variance</li>
 +
                        <li>L1_G: 0.1 T7 promoter - SarJ-UniRBS - TALEsp1- T7 terminator</li>
 +
                        <li>L1_H: 0.5 T7 promoter - RiboJ-UniRBS- Harmonized GFP - T7 terminator</li>
 +
                      </ul>
 +
                      <br>
 +
                      The following multiple transcriptional units, were assembled in MoClo level M backbones:
 +
                      <ul>
 +
                        <li>L1_C+L1_A </li>
 +
                        <li>L1_D+L1_B </li>
 +
                        <li>L1_D+L1_E </li>
 +
                        <li>L1_F+L1_H </li>
 +
                        <li>L1_G+L1_B </li>
 +
 
 +
                      </ul>
 +
                    </td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>Read more <a href="https://2019.igem.org/Team:TUDelft/Results">here</a>.</td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
              <div class="modal-footer">
 +
 
 +
              </div>
 +
            </div>
 +
 
 +
          </div>
 +
        </td>
 +
        <td rowspan=2 class="modalTD">
 +
          <!-- Trigger/Open The Modal -->
 +
          <button style="height:130px;" class="block modal-button" href="#myModal15">Translational Variantions</button>
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          <!-- The Modal -->
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 +
 
 +
            <!-- Modal content -->
 +
            <div class="modal-content">
 +
              <div class="modal-header">
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                <span class="close">×</span>
 +
                <h2>Translational Variantions</h2>
 +
              </div>
 +
              <div class="modal-body">
 +
                <table>
 +
                  <tr>
 +
                    <td>Goal:</td>
 +
                    <td>To demonstrate stable GFP expression despite change in translational rates
 +
 
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Protocols Used:</td>
 +
                    <td> <a href="https://2019.igem.org/Team:TUDelft/Experiments">Nanodrop DNA quantification</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Plasmid Isolation</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">MoClo</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Heat Shock Transformation</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Colony Polymerase PCR (GoTaq)</a>, <a href="https://2019.igem.org/Team:TUDelft/Experiments"><i>E. coli</i> Glycerol Stock</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">DNA Gel Electrophoresis</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Sequencing</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">Xgal Stock</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">OD600 Measurement</a>,
 +
                      <a href="https://2019.igem.org/Team:TUDelft/Experiments">FACS</a>
 +
 
 +
                    </td>
 +
                  </tr>
 +
                  <tr>
 +
                    <td>Plan:</td>
 +
                    <td>Gene expression independent of translational rates was demonstrated by cloning constructs in which the RBSs for TALE and GFP were switched out RBSs (BBa_B0032 and Universal RBS) of different strengths. The GFP expression was measured by FACS.
 +
                      To overcome translational variations associated with codon usage, eGFP sequence was predicted using our codon harmonizer tool. The function of harmonized eGFP was confirmed by detecting output fluorescence.
 +
 
 +
                    </td></tr>
 +
                  <tr>
 +
                    <td>Results:
 +
                    </td>
 +
                    <td>Read more <a href="https://2019.igem.org/Team:TUDelft/Results">here</a>.</td>
 +
                  </tr>
 +
                </table>
 +
              </div>
 +
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 +
 
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 +
            </div>
 +
 
 +
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 +
        </td>
 +
      </tr>
 +
 
 +
      <tr>
 +
        <td class="month">October</td>
 +
 
 +
      </tr>
 +
    </table>
 +
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 +
 
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Revision as of 07:33, 21 October 2019

Sci-Phi 29

Orthogonal Replication


Cloning Testing of Plasmid
April
May
June
July
August
September
October


Controllable Expression


Cloning Testing of Plasmid
April
May
June
July
August
September
October