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

Line 276: Line 276:
 
                     <img src="https://static.igem.org/mediawiki/2018/1/17/T--Valencia_UPV--comparison_GGBB_graphUPV2018.png">
 
                     <img src="https://static.igem.org/mediawiki/2018/1/17/T--Valencia_UPV--comparison_GGBB_graphUPV2018.png">
 
                   </a><h6 style="text-align: left; padding-left: 5em;">Comparison between fluorescence and absorbance of Golden Gate and BioBrick TU.</h6><div>
 
                   </a><h6 style="text-align: left; padding-left: 5em;">Comparison between fluorescence and absorbance of Golden Gate and BioBrick TU.</h6><div>
                   
 
                   
 
                   
 
 
                   
 
                   
 
                   
 
                   
 
 
 
             
 
                     
 
                   
 
 
                     <div>
 
                     <div>
                       
+
                         <div style="">
                       
+
                       
+
 
+
                       
+
               
+
 
+
                         <div style="
+
      ">
+
                       
+
 
+
                       
+
                 
+
 
+
            POSIBILIDAD DE HACER UN TEST ESTADÍSTICO O DAR ALGUN DATO QUE PRUEBE QUE SON IGUALES
+
                    FALTAN AÑADIR REFERENCIAS HARDWARE Y EXPERIMENTOS
+
 
           </div>
 
           </div>
 
 
               </div>
 
               </div>
             
 
 
      <!--
 
              <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:
 
 
        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:
 
                 
 
                  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.
 
 
            -->
 
 
 
             </div>
 
             </div>
 
         </div>
 
         </div>
Line 333: Line 285:
 
   </div>
 
   </div>
 
</section>
 
</section>
 
 
 
 
 
  
 
         <!--<div class="loader"></div>-->
 
         <!--<div class="loader"></div>-->

Revision as of 12:06, 12 October 2018

Stack Multipurpose HTML Template

Demonstrate

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:

  • 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:

    Test boards

    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 in order to implement the hot and cold zones, leading to the following design:

    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 which the drops will move. To prove the functioning of the thermocycler, an assembly of BBa_K2656024 (Link) into P10500 (Link) war made and the results are the following:

  • Hot and cold zones' experiment results
  • Once the assembling process has been made, it is necessary to electroporate the bacteria in order to do the transformation. 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.

    Electroporation experiment
  • 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 cultured tubes in which the bacteria will be stored. To those holders, there are sensors attached to monitor the growth of bacteria. To test the system, we have used the shaker while OD and Fluorescence measurements have been made, obtaining the following graphs:

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

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 sfGFP transcriptional unit that has been obtained by employing Golden Gate assembly method.

Assembly of BBa_K2656103 transformed into electrocompetent bacteria.

It has also been proven that by using linearized destination vectors in the Golden Gate assembly reactions, a very small number of bacteria transformed with vectors without the desired insert are obtained. Here you can see a GFP transcriptional unit obtained by using a linearized destination vector in the assembly reaction.

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 losing their competence.

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.

Finally, to compare the efficiency of Golden Gate vs BioBricks assembly, we have designed a comparative experiment 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.

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

CONTACT US igem.upv.2018@gmail.com