Difference between revisions of "Team:Valencia UPV/Demonstrate"

 
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                                <h4 style="
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                                    ">Index</h4>
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                                            ">Printeria concept-testing</a>
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                                            ">Printeria Wet Lab demonstrations</a>
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                                    </li>
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                              <a class="anchorOffset" id="Demonstrate"></a><p>
  
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                                After long months of effort and dedication, <b>Printeria</b>, the project of the <b>Valencia UPV team</b> is now a reality! All our work has been reflected in our device. With this project we intend to make the approach to Synthetic Biology a reality, but... to what extent has our team been able to develop Printeria? <b>Does it really work?</b>
  
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                                </p><a class="anchorOffset" id="concept-testing"></a><h4>Printeria concept-testing</h4>
  
              </section></div>
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                                <p>
<a class="anchorOffset" id="story"></a>
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                                Here we show all the evidence to prove that the Printeria device really works.
<section class="feature-large" style="padding-top: 6em; padding-bottom: 50px; outline: none;" data-scroll-id="story" tabindex="-1">
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                                  <li><p>
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                                    The <a href="https://2018.igem.org/Team:Valencia_UPV/Hardware#Entry" target="_blank"><b>entry of consumables system</b></a> is a completely original approach in order to dispense precise quantities of liquid in a compact and highly controllable way. In the following video it is shown how our entry system works:
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              <h4 style="
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                  ">Index</h4>
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                    <div class="tab__title" style="
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                        <a href="#concept-testing" class="lateral inner-link" style="
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                          ">Printeria device concept-testing</a>
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                          ">Printeria Wet Lab demonstrations</a>
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              <a class="anchorOffset" id="Demonstrate"></a>
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              <h2 class="h2Demonstrate">Demonstrate</h2>
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              <p>
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              After long months of effort and dedication, the <b>Printeria project of the Valencia UPV iGEM 2018 team is now a reality!</b> All our work has been reflected in our Printeria device. With this project we intend to make the approach to Synthetic Biology a reality, but... to what extent has our team been able to develop Printeria? Does it really work? <b>Let's see all the evidences of our achievements...</b>
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              </p>
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              <a class="anchorOffset" id="concept-testing"></a>
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                                  <a href="https://static.igem.org/mediawiki/2018/d/db/T--Valencia_UPV--ENTRADA5UPV2018.gif" data-lightbox="true">
              <h4>Printeria device concept-testing</h4>
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                                    <img src="https://static.igem.org/mediawiki/2018/d/db/T--Valencia_UPV--ENTRADA5UPV2018.gif">
              <p>
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                                  </a>
              <b>Here we show you all the evidence to convince you that the Printeria device really works...</b>
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                                  <h6 style="text-align: right;">Image 1.Proof of concept of the entry system</h6>
              </p>
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              <ul>
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          <li><p>
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          The  is a completely original approach in order to dispense precise quantities of liquid in a compact and highly controllable way. You can check in this video how our device input system works...
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          </p></li>
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                <a href="https://static.igem.org/mediawiki/2018/d/db/T--Valencia_UPV--ENTRADA5UPV2018.gif" data-lightbox="true">
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                                  <li><p>
                    <img src="https://static.igem.org/mediawiki/2018/d/db/T--Valencia_UPV--ENTRADA5UPV2018.gif" />
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                                    The <b>digital microfluidics technology</b> is the basis of Printeria. It allows us to recreate the experiments that take place in the lab in an accurate and automated way, providing a high<a href="https://2018.igem.org/Team:Valencia_UPV/Hardware#Reaction" target="_blank"> control of the reactions</a>. First, we accomplished the movement of the droplets in <b>smaller test boards</b>, as you can see here:
                </a>
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                <h6 style="text-align: left; padding-left: 5em;">Proof of concept of the input system.</h6>
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          <li><p>
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          The <b>digital MicroFluidics</b> is at the centre of Printeria, and it enables a high <a href="https://2018.igem.org/Team:Valencia_UPV/Hardware#Reaction" target="_blank">control of the reactions</a>. We have accomplished the movement on the droplets in smaller test boards, as you can see here:
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          </p>
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          <p>
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                                    </p>
          And then we moved to a more clean product that is able to hold a larger number of pads and it is easier to control. You can see that result below:
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                                    <a href="https://static.igem.org/mediawiki/2018/8/88/T--Valencia_UPV--PCBPRUEBAUPV2018.jpeg" data-lightbox="true">
          </p>
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                                    <img src="https://static.igem.org/mediawiki/2018/8/88/T--Valencia_UPV--PCBPRUEBAUPV2018.jpeg">
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                                  </a>
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                                  <h6 style="text-align: right;">Image 2. Test board</h6>
  
          </p></li>
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                                    <p>
          VIDEOS
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                                    After testing the digital microfluidic technology, we were able to approach the <a href="https://2018.igem.org/Team:Valencia_UPV/Hardware#finaldesign" target="_blank"><b>final surface design</b></a>. We made it larger so that it could hold a <b>higher number of pads</b> and,  the hot and cold zones could be implemented, leading to the following design:
          <li><p>
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                                    </p>
          The droplet needs to be heated and cooled for the reaction to take place. We have implemented these <b>hot and cold zones via a resistor and peltiers</b>. Here the stability of the temperature control for both is demonstrated.
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          </p></li>
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          GRÁFICA
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          <li><p>
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          We have included the design of out <b>electroporator</b> from <a href="https://2016.igem.org/Team:Valencia_UPV" target="_blank" style="padding-right: 0">Hype It iGEM project</a>, developed in 2016 by Valencia UPV team. The results of our experiment using it are shown here:
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          </p></li>
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          GRÁFICA
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          <li><p>
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          For the output we have included an <b>Optical Density and Fluorescence sensors</b> in order to measure data from the bacteria that are growing on the <a href="https://2018.igem.org/Team:Valencia_UPV/Hardware#Measurement" target="_blank">measurement zone</a>. The results of our tests are the following:
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          <a href="https://static.igem.org/mediawiki/2018/5/5a/T--Valencia_UPV--od_Printeria_sensorUPV2018.png" data-lightbox="true">
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                                    <a href="https://static.igem.org/mediawiki/2018/a/ab/T--Valencia_UPV--PCBFINALUPV2018.jpeg" data-lightbox="true">
      <img src="https://static.igem.org/mediawiki/2018/5/5a/T--Valencia_UPV--od_Printeria_sensorUPV2018.png" />
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                                    <img src="https://static.igem.org/mediawiki/2018/a/ab/T--Valencia_UPV--PCBFINALUPV2018.jpeg">
      </a>
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                                  </a>
      <h6 style="text-align: left; padding-left: 5em;">OD measurements registred by Printeria OD sensor adn fitted to an exponential curve of cell growth.</h6>
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                                  <h6 style="text-align: right; ">Image 3. PCB surface final design</h6>
      GRAFICA FLUORESCENCIA
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                                  </li>
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                                  <li><p>
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                                    In order to do the assembly, it is necessary to make the droplets go through <a href="https://2018.igem.org/Team:Valencia_UPV/Hardware#thermo" target="_blank"><b>cyclic temperature changes</b></a>. For this purpose, cold and hot zones have been implemented, thanks to a resistance and two peltiers between the drops will move. To prove if the zones work, an assembly of <a href="http://parts.igem.org/Part:BBa_K2656103" target="_blank"><b>BBa_K2656024</b></a> into <a href="http://parts.igem.org/Part:BBa_P10500" target="_blank"><b>BBa_P10500</b></a> was made  with good results:
 +
                                  </p></li>
  
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                                  <a href="https://static.igem.org/mediawiki/2018/9/9d/T--Valencia_UPV--PETRIUPV2018.jpeg" data-lightbox="true">
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                                    <img src="https://static.igem.org/mediawiki/2018/9/9d/T--Valencia_UPV--PETRIUPV2018.jpeg">
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                                          "><h6>Image 4. Hot and cold zones' experiment results</h6>
 +
                                        </div></a>
  
<a class="anchorOffset" id="Wetlab_dem"></a>
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            <li>
              <h4>Printeria Wet Lab demonstrations</h4>  
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                                    <p>Once the assembly process has been made, it is necessary to do the transformation of bacteria. We have implemented an <a href="https://2018.igem.org/Team:Valencia_UPV/Hardware#electroporator" target="_blank"><b>electroporator</b></a> based on the design made by the <a href="https://2016.igem.org/Team:Valencia_UPV/Hardware" target="_blank" style="padding-right: 0">Hype It iGEM project (Valencia_UPV 2016 team)</a>. The electroporator was tested in a cuvette before implementing it in Printeria.</p>
              <p>
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                                    <div>
              <b>In this section you can check all our Wet Lab demonstrations...</b>
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              We have proved that assembling functional composite parts with the <a href="" target="_blank"> Golden Gate method</a> with our basic parts is possible... Here you can see a <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2656103" target="_blank">sfGFP transcriptional unit</a> that has been obtained by employing Golden Gate assembly method.
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                                          <a href="https://static.igem.org/mediawiki/2018/e/eb/T--Valencia_UPV--ELECTROPORADOR1UPV2018.jpeg" data-lightbox="true">
  
          <a href="https://static.igem.org/mediawiki/2018/b/b3/T--Valencia_UPV--BBa_K2656103_imgUPV2018.png" data-lightbox="true">
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                                          <img src="https://static.igem.org/mediawiki/2018/e/eb/T--Valencia_UPV--ELECTROPORADOR1UPV2018.jpeg" style="
<img src="https://static.igem.org/mediawiki/2018/b/b3/T--Valencia_UPV--BBa_K2656103_imgUPV2018.png" />
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                                          margin-top: 0.5em;">
</a>
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<h6 style="text-align: left; padding-left: 5em;">Assembly of BBa_K2656103 transformed into electrocompetent bacteria.</h6>
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              </li>
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                                          </a>
              <li><p>
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              It has also been proven that by using <b>linearised target vectors</b>, a very small number of bacteria transformed with vectors without the desired insert are obtained. Here you can see a <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2656105" target="_blank">GFP TU</a> obtained by using a linearised target vector.
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              </p>
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              <a href="https://2018.igem.org/File:T--Valencia_UPV--BBa_K2656105_imgUPV2018.png" data-lightbox="true">
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                                        </div>
<img src="https://static.igem.org/mediawiki/2018/0/0c/T--Valencia_UPV--BBa_K2656105_imgUPV2018.png" />
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<h6 style="text-align: left; padding-left: 5em;">Transformed bacteria resulting from the assembly of BBa_K2656105 TU.</h6>
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                                          "><h6>Image 5a. Electroporator set up.<br>Image 5b. Results of the electroporator testing.</h6>
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                                  <a id="curvesFOD" class="anchorOffset"></a>
              Another demonstration is that electrocompetent bacteria can be stored at -20ºC for at least two weeks and rested for 4 hours at room temperature (30.3ºC) without losing their competence.
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                                    Once the transformed bacteria are ready, they are sent to the <a href="https://2018.igem.org/Team:Valencia_UPV/Hardware#Measurement" target="_blank">measurement system</a> in order to culture them while <b>OD and fluorescence measurements</b> are taken. It consists of a system based on a shaker, which has holders for the incubated tubes in which the bacteria will be stored. To those holders, there are sensors attached to monitor the growth of the bacteria and to measure the fluorescence obtained. To test the system, we have used the shaker while OD and Fluorescence measurements have been made. The results of our tests are the following:
<img src="https://static.igem.org/mediawiki/2018/e/ea/T--Valencia_UPV--competent_cellsUPV2018.png" />
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<h6 style="text-align: left; padding-left: 5em;">Electrocompetent bacteria at 1:100 (left), 1:10 (center) and 1:1 (right) dilution stored at -20°C for two weeks and set at Room Temperature (37°C) 4 hours before electroporation transformed with a control plasmid.</h6>
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                                  <a href="https://static.igem.org/mediawiki/2018/5/5a/T--Valencia_UPV--od_Printeria_sensorUPV2018.png" data-lightbox="true">
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            Finally, to compare the efficiency of <b>Golden Gate vs BioBricks assembly</b>, we have designed a <a href="" target="_blank">comparative experiment</a> between two TU of identical structure but assembled with Golden Gate and BioBricks method, respectively. Our results demonstrate that both methods have the same efficiency.
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                                  </a>
            </p>
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                                  </p><h6 style="text-align: right; ">Figure 1. OD measurements registred by Printeria's OD sensor and fitted to an exponential curve of cell growth.</h6>
  
            <a href="https://static.igem.org/mediawiki/2018/1/17/T--Valencia_UPV--comparison_GGBB_graphUPV2018.png" data-lightbox="true">
 
<img src="https://static.igem.org/mediawiki/2018/1/17/T--Valencia_UPV--comparison_GGBB_graphUPV2018.png" />
 
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<h6 style="text-align: left; padding-left: 5em;">Comparison between fluorescence and absorbance of Golden Gate and BioBrick TU.</h6>
 
  
POSIBILIDAD DE HACER UN TEST ESTADÍSTICO O DAR ALGUN DATO QUE PRUEBE QUE SON IGUALES
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            FALTAN AÑADIR REFERENCIAS HARDWARE Y EXPERIMENTOS
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                                  </p><h6 style="text-align: right; ">Figure 2. OD calibration curve. We have calibrated the data from the OD Printeria sensor with the OD data from the Biotek Cytation3 equipment.</h6>
  
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                                  </a>
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                                  <h6 style="text-align: right; ">Figure 3. Fluorescence calibration curve. We have calibrated the data from the Fluorescence Printeria sensor with the fluorescence data from the Biotek Cytation3 equipment.</h6>
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            <p>
 
              One of the Printeria Wetlab challenges during the project has been to demonstrate that Printeria transcriptional units (TU) obtained with <a href="" target="_blank">Golden Gate assembly technology</a> are as efficients as TU assembled by BioBrick technology. The main advantage offered by Golden Gate technology is the posibility to perform an assembly reaction with many parts in a single step. This is the main reason why we have design an extensive <a href="https://2018.igem.org/Team:Valencia_UPV/Part_Collection" target="_blank">Part Collection</a> standarized to GoldenBraid 3.0 grammar. However, <b>which is the efficiency of these parts compared to BioBrick parts?</b>
 
              In this way we can confirm, thanks to the results obtained, that <b>the efficiency will be the same independently of the assembly method</b>.
 
            </p>
 
            <p>
 
              <b>For the correct working of Printeria many technologies have to interact and work together</b> in order to produce the desired reaction. But the individual parts themselves also have to work properly in order for the whole to function. In this way, we want to <b>our individual proofs of concept</b> that then are joined on Printeria so that finally our user does not have to think about the complex processes that happen inside Printeria.
 
              </p>
 
  
              The <b>Input system</b> is a completely original approach in order to dispense precise quantities of liquid in a compact and highly controllable way. As well as to make it easier for the user to introduce the materials all at once or individually (discover more about the inner workings of <a href="" target="_blank">Printeria input system</a>). You can see our concept test on this video:
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The <b>digital MicroFluidics</b> is at the centre of Printeria, and it enables a high <a href="" target="_blank">control of the reactions</a>. We have accomplished the movement on the droplets in smaller test boards, as you can see here:
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                                </ul><a class="anchorOffset" id="Wetlab_dem"></a><h4>Printeria Wet Lab demonstrations</h4>
          The droplet needs to be heated and cooled for the reaction to take place. We have implemented these <b>hot and cold zones</b> via a <a href="" target="_blank">resistor and a Peltier</a>. Here the stability of the temperature control for both is demonstrated.
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                                In this section you can check all our Wet Lab demonstrations:
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                              We have proved that assembling functional composite parts with the <a href="https://2018.igem.org/Team:Valencia_UPV/Design#GB" target="_blank"> Golden Gate method</a> with our basic parts is possible. Here you can see a <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2656109" target="_blank">mRFP transcriptional unit</a> been obtained with the Golden Gate assembly method.
 +
                                 
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                                    Red colonies are these that have correctly taken up the recombinant plasmid (which carries the composite part), whereas white colonies are those carrying the non-recombinant plasmid. As it can be seen, transcriptional unit assembly has been effectively achieved with our DNA basic parts. On the other hand, the percentage of recombinant colonies is high, but there are still more colonies with the not desired plasmid.
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            <h6 style="
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            Thus, we also desired to avoid the plate colony screening step. To do so, we have used the linearized destination vector for the Level 1 Golden Gate assembly. In this way, only the circularized plasmids will be taken up by the bacteria with high efficiency, as linear double-stranded DNA transformation in E. coli is generally inefficient due to the exonucleolytic degradation. Thus, we have proved that using the linearized destination vector very small number of bacteria transformed with the vector without the insert are obtained. Here you can see a GFP transcriptional unit obtained with 25 ng of linearized destination vector. For both experiments (this and the previous one), 10G electrocompetent cells with same transformation efficiency had been used, and transforming was made with same DNA quantity. Consequently, it can be concluded that the efficiency of the reaction is lower compared to the one with the circularized plasmid (as there are less transformants in the plate), but the number of plasmids without the desired insert is highly reduced too (6 colonies with the non-recombinant vector / 71 total colonies = 8.5 % background colonies). For Printeria use, the efficiency of this transformation is high enough.
  
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            <h6>Image 7. TU assembly of K2656105 with linearized plasmid </h6></div>
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                                      Another demonstration is that electrocompetent bacteria can be stored at -20ºC for at least two weeks and chilled for 4 hours at room temperature (30.3ºC) without completely losing their competence.
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                                    </p><a href="https://static.igem.org/mediawiki/2018/e/ea/T--Valencia_UPV--competent_cellsUPV2018.png" data-lightbox="true">
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                            </a><h6 style="
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                  margin-right: 0px !important;text-align: left; padding-left: 0em;">Image 8. Serial decimal dilutions (from left to right: 10<sup>-2</sup>, 10<sup>-1</sup> and 10<sup>0</sup>) of transformed electrocompetent bacteria stored at -20°C for two weeks and set at Room Temperature (37°C) 4 hours before electroporation. These bacteria are transformed with P10500 and plated in LB-agar petri dishes with cloranphenicol. </h6></div><div style="
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                  </p><p>The transformation efficiency was 10<sup>4</sup> CFU/microgram of DNA. This low data may be due to the fact that instead of using 1ng of plasmid control, 200 were used, and efficiency may be lost. </p><p>This efficiency may be low for a normal cloning process, but thanks to avoiding the plate screening phase, we do not require higher efficiencies.</p>
 +
                              <p>Finally, to compare the efficiency of <b>Golden Gate vs BioBricks assembly</b>, we have designed a <a href="https://2018.igem.org/Team:Valencia_UPV/Experiments#GGBB_exp" target="_blank">comparative experiment</a> between two transcriptional units of identical structure but assembled with Golden Gate and BioBricks method, respectively. Our results demonstrate that both methods have the same efficiency.</p>
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                                    <p></p></div><a href="https://static.igem.org/mediawiki/2018/1/17/T--Valencia_UPV--comparison_GGBB_graphUPV2018.png" data-lightbox="true">
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                              </a><h6 style="text-align: left; padding-left: 5em;">Figure 4. Comparison between fluorescence and absorbance of Golden Gate and BioBrick TU.</h6><div>
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Latest revision as of 11:11, 7 December 2018

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After long months of effort and dedication, Printeria, the project of the Valencia UPV team is now a reality! All our work has been reflected in our device. With this project we intend to make the approach to Synthetic Biology a reality, but... to what extent has our team been able to develop Printeria? Does it really work?

Printeria concept-testing

Here we show all the evidence to prove that the Printeria device really works.

  • The entry of consumables system is a completely original approach in order to dispense precise quantities of liquid in a compact and highly controllable way. In the following video it is shown how our entry system works:

  • Image 1.Proof of concept of the entry system
  • The digital microfluidics technology is the basis of Printeria. It allows us to recreate the experiments that take place in the lab in an accurate and automated way, providing a high control of the reactions. First, we accomplished the movement of the droplets in smaller test boards, as you can see here:

    Image 2. Test board

    After testing the digital microfluidic technology, we were able to approach the final surface design. We made it larger so that it could hold a higher number of pads and, the hot and cold zones could be implemented, leading to the following design:

    Image 3. PCB surface final design
  • In order to do the assembly, it is necessary to make the droplets go through cyclic temperature changes. For this purpose, cold and hot zones have been implemented, thanks to a resistance and two peltiers between the drops will move. To prove if the zones work, an assembly of BBa_K2656024 into BBa_P10500 was made with good results:

  • Image 4. Hot and cold zones' experiment results
  • Once the assembly process has been made, it is necessary to do the transformation of bacteria. We have implemented an electroporator based on the design made by the Hype It iGEM project (Valencia_UPV 2016 team). The electroporator was tested in a cuvette before implementing it in Printeria.

    Image 5a. Electroporator set up.
    Image 5b. Results of the electroporator testing.
  • Once the transformed bacteria are ready, they are sent to the measurement system in order to culture them while OD and fluorescence measurements are taken. It consists of a system based on a shaker, which has holders for the incubated tubes in which the bacteria will be stored. To those holders, there are sensors attached to monitor the growth of the bacteria and to measure the fluorescence obtained. To test the system, we have used the shaker while OD and Fluorescence measurements have been made. The results of our tests are the following:

    Figure 1. OD measurements registred by Printeria's OD sensor and fitted to an exponential curve of cell growth.

    Figure 2. OD calibration curve. We have calibrated the data from the OD Printeria sensor with the OD data from the Biotek Cytation3 equipment.
    Figure 3. Fluorescence calibration curve. We have calibrated the data from the Fluorescence Printeria sensor with the fluorescence data from the Biotek Cytation3 equipment.

Printeria Wet Lab demonstrations

In this section you can check all our Wet Lab demonstrations:

We have proved that assembling functional composite parts with the Golden Gate method with our basic parts is possible. Here you can see a mRFP transcriptional unit been obtained with the Golden Gate assembly method. Red colonies are these that have correctly taken up the recombinant plasmid (which carries the composite part), whereas white colonies are those carrying the non-recombinant plasmid. As it can be seen, transcriptional unit assembly has been effectively achieved with our DNA basic parts. On the other hand, the percentage of recombinant colonies is high, but there are still more colonies with the not desired plasmid.

Image 6. Transformed bacteria resulting from the assembly of BBa_K2656109 TU.

Thus, we also desired to avoid the plate colony screening step. To do so, we have used the linearized destination vector for the Level 1 Golden Gate assembly. In this way, only the circularized plasmids will be taken up by the bacteria with high efficiency, as linear double-stranded DNA transformation in E. coli is generally inefficient due to the exonucleolytic degradation. Thus, we have proved that using the linearized destination vector very small number of bacteria transformed with the vector without the insert are obtained. Here you can see a GFP transcriptional unit obtained with 25 ng of linearized destination vector. For both experiments (this and the previous one), 10G electrocompetent cells with same transformation efficiency had been used, and transforming was made with same DNA quantity. Consequently, it can be concluded that the efficiency of the reaction is lower compared to the one with the circularized plasmid (as there are less transformants in the plate), but the number of plasmids without the desired insert is highly reduced too (6 colonies with the non-recombinant vector / 71 total colonies = 8.5 % background colonies). For Printeria use, the efficiency of this transformation is high enough.

Image 7. TU assembly of K2656105 with linearized plasmid

Another demonstration is that electrocompetent bacteria can be stored at -20ºC for at least two weeks and chilled for 4 hours at room temperature (30.3ºC) without completely losing their competence.

Image 8. Serial decimal dilutions (from left to right: 10-2, 10-1 and 100) of transformed electrocompetent bacteria stored at -20°C for two weeks and set at Room Temperature (37°C) 4 hours before electroporation. These bacteria are transformed with P10500 and plated in LB-agar petri dishes with cloranphenicol.

The transformation efficiency was 104 CFU/microgram of DNA. This low data may be due to the fact that instead of using 1ng of plasmid control, 200 were used, and efficiency may be lost.

This efficiency may be low for a normal cloning process, but thanks to avoiding the plate screening phase, we do not require higher efficiencies.

Finally, to compare the efficiency of Golden Gate vs BioBricks assembly, we have designed a comparative experiment between two transcriptional units of identical structure but assembled with Golden Gate and BioBricks method, respectively. Our results demonstrate that both methods have the same efficiency.

Figure 4. Comparison between fluorescence and absorbance of Golden Gate and BioBrick TU.

CONTACT US igem.upv.2018@gmail.com