Difference between revisions of "Team:Ionis Paris"

 
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    <link rel="stylesheet" type="text/css" href="https://2016.igem.org/Template:IONIS_Paris-style-css-template?action=raw&ctype=text/css" />
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    <!-- ====HEADER PARALUX==== -->
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        <link rel="stylesheet" type="text/css" href="https://2016.igem.org/Template:IONIS_Paris-test?action=raw&ctype=text/css" />
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    <section id="ancre" class="header_paralux" style="background: url('https://static.igem.org/mediawiki/2016/8/88/HOME.jpg');  background-size: cover;
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    background-repeat: no-repeat;
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    position: relative;
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        <!-- ====BREADCUM START==== -->  
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    background-attachment: fixed;">
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         <section class="blog_banner_ecoli">
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                             <h1 id="back_to_the_top">Proof of Concept</h1>
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                             <div class="page_down text-center">
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                                <a href="#quantifly" class="go-down">
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                                    <i class="zmdi zmdi-long-arrow-down testi_prev"></i>
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                                </a>
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         </div>
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        <!-- ====BREADCUM END==== -->
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    </section>
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    <!-- ====HEADER PARALUX END==== -->
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      <!-- ====START BLOG TABLE==== -->
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        <section id="about" class="about_area section-padding">
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                                    <h2 class="secHd">Toluene evaporation test</h2>
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    <!--  <section class="feature_area" >
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                      <div class="single_feature text-center">
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                          <i class="zmdi zmdi-devices"></i>
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                          <h4>Branding</h4>
 +
                          <div class="single_cap">
 +
                          <p>As a business owner or manager, the decision to opt faor offshore software can be difficult.</p>
 +
                          <a class="th_bt btn waves-effect" href="#!">Read More</a>
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                          </div>
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                      </div>
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                  </div>
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                  <div class="col-xs-12 col-sm-6 col-md-3 fix_p">
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                      <div class="single_feature text-center">
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                          <i class="zmdi zmdi-globe-alt"></i>
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                          <h4>Web Development</h4>
 +
                          <div class="single_cap">
 +
                          <p>As a bu siness owner or manager, the decision to opt faor offshore software can be difficult.</p>
 +
                          <a class="th_bt btn waves-effect" href="#!">Read More</a>
 +
                          </div>
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                      </div>
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 +
                  <div class="col-xs-12 col-sm-6 col-md-3 fix_p">
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                      <div class="single_feature text-center">
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                          <i class="zmdi zmdi-apple"></i>
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                          <h4>Mobile Application</h4>
 +
                          <div class="single_cap">
 +
                          <p>As a bu siness owner or manager, the decision to opt faor offshore software can be difficult.</p>
 +
                          <a class="th_bt btn waves-effect" href="#!">Read More</a>
 +
                          </div>
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                      </div>
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                  </div>
 +
                  <div class="col-xs-12 col-sm-6 col-md-3 fix_p">
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                      <div class="single_feature text-center">
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                          <i class="zmdi zmdi-camera"></i>
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                          <h4>Photography</h4>
 +
                          <div class="single_cap">
 +
                          <p>As a bu siness owner or manager, the decision to opt faor offshore software can be difficult.</p>
 +
                          <a class="th_bt btn waves-effect" href="#!">Read More</a>
 +
                          </div>
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                      </div>
 +
                  </div>
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              </div>
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          </div>
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      </section> -->
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    <!-- ====FEATURE AERE END==== -->
  
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    <!-- ====ABOUT US==== -->
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    <section id="about" class="about_area section-padding">
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        <div class="container">
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            <div class="row">
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                <div class="col-xs-12">
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                    <div class="section_title mb80">
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                        <h5 id="qantifly" class="smallHd text-center">What is
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                        </h5>
 +
                        <h2 class="secHd text-center">Quantifly ?</h2>
 +
                    </div>
 +
                </div>
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            </div>
  
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              <h3><font color = "A2233C">Protocol</font> </h3>
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            <div class="col-xs-12 col-sm-6">
 +
                <div class="about_left">
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                    <img src="https://static.igem.org/mediawiki/2016/2/23/Ville.png" alt="" />
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                </div>
 +
            </div>
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            <div class="col-xs-12 col-sm-6">
 +
                <div class="section_title">
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                    <h5 class="smallHd">(VOC)</h5>
 +
                    <h2 class="secHd">VOLATILE ORGANIC COMPOUNDS</h2>
 +
                </div>
 +
                <div class="about_text">
 +
                    <p>Nowadays, Volatile Organic Compounds (VOCs) are commonly found pollutants that have been proven
 +
                        present in a wide variety of everyday life situations. Though they are dangerous and present
 +
                        almost everywhere, VOCs are not efficiently detected. Usual detection devices are very unprecise
 +
                        or require a very long exposure time.</p>
  
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  <p>The toluene is known to be a volatile compound. This is why we decided to determine its rate of evaporation before proceeding to any subsequent manipulation with toluene. This will determine the care taken when handling it as well as the time the stock solution will be kept. <br/>
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                </div>
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1mL of pure toluene (867g/L, 244511 Sigma-Aldrich) was added  in a 1.5mL Eppendorf tube. The tube was left open under a chemical hood and its weight was calculate every 30 minutes. </p>
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            </div>
 +
        </div>
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        </div>
 +
    </section>
 +
    <!-- ====ABOUT US==== -->
 +
    <!-- ====ABOUT US==== -->
 +
    <section id="about" class="about_area section-padding">
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        <div class="container">
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            <div class="row">
 +
                <div class="col-xs-12 col-sm-6">
 +
                    <div class="section_title">
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                        <h5 class="smallHd">Bacteria</h5>
 +
                        <h2 class="secHd">Biosensor</h2>
 +
                    </div>
 +
                    <div class="about_text">
 +
                        <p>This is why we developed the Quantifly project : a combination of a bacterial biosensor,
 +
                            perfectly adapted to on-field measurements, and an innovative drone that will allow us to
 +
                            easily and rapidly quantify the amount of VOCs in the air. The biosensor relies on
 +
                            bioluminescence, a biochemical phenomenon that is commonly used in cell imaging. We are
 +
                            trying to develop a new application for bioluminescence that should allow us to create a
 +
                            biosensor of a new kind, entirely different from the existing devices.</p>
 +
                     
 +
                    </div>
 +
                </div>
 +
                <div class="col-xs-12 col-sm-6">
  
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              <h3><font color = "A2233C">Results</font></h3>
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    <div class="about_left">
 +
                    <img src="https://static.igem.org/mediawiki/2016/7/75/T--IONIS_Paris--Biosensor.png" alt="" />
 +
                </div>
 +
                </div>
 +
         
  
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  <center><table>
 
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            <tr>
 
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              <td><p>Time</p></td>
 
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              <td><p> Weight</p></td>
 
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            </tr>
 
  
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             <tr>
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             </div>
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              <td><p> 1h</p> </td>
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        </div>
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              <td><p> Weight</p> </td>
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    </section>
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            </tr>
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    <!-- ====ABOUT US==== -->
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    <!-- ====ABOUT US==== -->
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    <section id="about" class="about_area section-padding">
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        <div class="container">
 +
            <div class="row">
 +
                <div class="col-xs-12 col-sm-6">
 +
                    <div class="about_left">
 +
                        <img src="https://static.igem.org/mediawiki/2016/f/f5/Drone_1.png" alt="drone" />
 +
                    </div>
 +
                </div>
 +
                <div class="col-xs-12 col-sm-5">
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                    <div class="section_title">
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                        <h5 class="smallHd">Bacteria</h5>
 +
                        <h2 class="secHd">Drone</h2>
 +
                    </div>
 +
                    <div class="about_text">
 +
                        <p>Concerning the drone, it is designed to safely contain our organisms and carry them on the
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                            field, thus acting as a mobile detection platform.
 +
                        </p>
  
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             <tr>
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                    </div>
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              <td><p> 1h30</p> </td>
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                </div>
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              <td><p> Weight</p> </td>
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             </div>
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             </tr>
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        </div>
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    </section>
 +
    <!-- ====ABOUT US==== -->
 +
    <!-- ====START SKILL AND VEDIO TABLE==== -->
 +
    <section class="skill_and_video">
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        <div class="video_area">
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            <div class="video_control">
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                <div class="dtable">
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                    <div class="dcell">
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                        <a href="https://www.youtube.com/watch?v=G0SF4aWuL4E" data-type="YouTube" id="control"
 +
                          class="btn waves-effect waves-light veno">
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                            <i class="zmdi zmdi-play"></i>
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                        </a>
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                        <h4>Check out the Video</h4>
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                    </div>
 +
                </div>
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             </div>
 +
        </div>
  
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            <tr>
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        <div class="row">
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              <td><p> 2h</p> </td>
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        <div class="col-sm-12">
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              <td><p> Weight</p> </td>
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<center><video width="760" height="415"  poster="https://static.igem.org/mediawiki/2015/8/83/Logo_IONIS.png" controls><source src="https://static.igem.org/mediawiki/2016/0/0e/T--Ionis_Paris--QuantiflyProjectVideo.mp4" type='video/mp4'/><a href="https://www.youtube.com/watch?v=G0SF4aWuL4E"><img border="0" src="https://static.igem.org/mediawiki/2015/8/83/Logo_IONIS.png" alt="Click to view on Youtube" width="760" height="415"></a>
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            </tr>
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<p><font color ="DeepPink">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</font></p></video></center>
  
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             <tr>
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             </div>
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              <td><p> 2h30</p>  </td>
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        </div>
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              <td><p> Weight</p> </td>
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            </tr>
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          </table></center>
 
  
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  <p> As shown in the table above, after 6 hours the weight decrease ……. compared to the starting weight. <br/>
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        </div>
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With this result, we can next test the lethality of <i>E.Coli</i> DH5α when exposed to several toluene concentrations. Moreover, those results ensure that the toluene will not evaporate from the liquid medium during an incubation time of 6 hours or less.<br/>
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        </div>
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The stock solution of toluene will be prepare again for each replicates.
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        </div>
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</p>
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        </div>
 +
    </section>
 +
    <!-- ====END SKILL AND VEDIO TABLE==== -->
 +
<!-- ====ABOUT US==== -->
 +
        <section id="about" class="about_area section-padding">
 +
            <div class="container">
 +
                <div class="row">
 +
                    <div class="col-xs-12 col-sm-6">
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                        <div class="about_left">
 +
                            <img src="https://static.igem.org/mediawiki/2016/b/ba/Ionis_page_gold_medal.jpg" alt=""/>
 +
                        </div>
 +
                    </div>
 +
                    <div class="col-xs-12 col-sm-6">
 +
                        <div class="section_title">
 +
                            <h5 class="smallHd">What we achieved</h5>
 +
                            <h2 class="secHd">About our Successes</h2>
 +
                        </div>
 +
                        <div class="about_text">
 +
                        <p class="ab_text">This year, we won a Gold Medal for our Quantifly project and were nominated for the following prizes : «<a href = "https://2016.igem.org/Team:Ionis_Paris/Description"><font color = DeepPink>Best Environmental Project</font></a>» , «<a href = "https://2016.igem.org/Team:Ionis_Paris/Design"><font color = DeepPink>Best Applied Design</font></a>» and «<a href = "https://2016.igem.org/Files/presentation/Ionis_Paris.pdf"><font color = DeepPink>Best Presentation</font></a>».</p>
 +
                            <a class="th_bt btn waves-effect waves-light" href="https://2016.igem.org/Team:Ionis_Paris/Prices">Read More</a>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
        </section>
 +
        <!-- ====ABOUT US==== -->
  
  
 +
    <!-- ====START OUR TEAM AREA==== -->
 +
    <section class="our_team">
 +
        <div class="container">
 +
            <div class="row">
 +
                <div class="col-xs-12">
 +
                    <div class="section_title mb80">
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                        <h5 class="smallHd text-center">OUR BEST INTER-TEAM
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                        </h5>
 +
                        <h2 class="secHd text-center">COLLABORATION</h2>
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                    </div>
 +
                </div>
 +
            </div>
 +
            <div class="row team_details_area">
 +
                <div class="col-xs-12 col-sm-6 fix_p">
 +
                    <div class="team_content">
 +
                        <div class="team_cotent_slid">
  
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                                     <h2 class="secHd"> Survival test to toluene </h2>
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                            <div class="item">
 +
                                <h1>The European <br>Experience </h1>
 +
                                <h6>In Paris 2/07/2016</h6>
 +
                                <p>The iGEM IONIS team and the iGEM Evry team worked together to accomplish a great gathering of the European teams: an European Jamboree! We organized over a week-end a symposium with more than 150 people that could assist to conferences and expose their projects, and create great network through the iGEM competition. We also wanted to give all European teams the opportunity to live an incredible week-end, by coming to Paris and being able to live the “science dream” in the city of light!
 +
                                </p>
 +
                                <ul class="team_social">
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                                     <li>
 +
                                        <a href="https://www.facebook.com/The.European.Experience.2016/?fref=ts">
 +
                                            <i class="zmdi zmdi-facebook btn waves-effect"></i>
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                                        </a>
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                                    </li>
 +
                                    <li>
 +
                                        <a href="https://twitter.com/igem_ionis">
 +
                                            <i class="zmdi zmdi-twitter btn waves-effect"></i>
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                                        </a>
 +
                                    </li>
 +
                                 
 +
                                </ul>
 +
                                <a class="th_bt btn waves-effect waves-light" href="https://2016.igem.org/Team:Ionis_Paris/Events">Learn More
 +
                                    <i class="zmdi zmdi-long-arrow-right"></i>
 +
                                </a>
 +
                            </div>
 +
                            <div class="item">
 +
                                <h1>A professional...</h1>
 +
                                <h6>V.I.Ps welcome</h6>
 +
                                <p>We organized some conferences, followed by a cocktail on Paris' rooftops! We even had the luck to count Mr. RETTBERG among our guests.</p>
 +
                                <ul class="team_social">
 +
                                    <li>
 +
                                        <a href="https://www.facebook.com/The.European.Experience.2016/?fref=ts">
 +
                                            <i class="zmdi zmdi-facebook btn waves-effect"></i>
 +
                                        </a>
 +
                                    </li>
 +
                                    <li>
 +
                                        <a href="https://twitter.com/igem_ionis">
 +
                                            <i class="zmdi zmdi-twitter btn waves-effect"></i>
 +
                                        </a>
 +
                                    </li>
 +
             
 +
                                </ul>
 +
                                <a class="th_bt btn waves-effect waves-light" href="https://2016.igem.org/Team:Ionis_Paris/Events">Learn More
 +
                                    <i class="zmdi zmdi-long-arrow-right"></i>
 +
                                </a>
 +
                            </div>
 +
                            <div class="item">
 +
                                <h1>...and international event</h1>
 +
                                <h6>organized by iGEM IONIS and iGEM Evry</h6>
 +
                                <p>Our event gathered 170 iGEMers, from 35 idifferent teams scattered in 11 countries! We had tons of fun with everyone and discovered amazing projects.</p>
 +
                                <ul class="team_social">
 +
                                    <li>
 +
                                        <a href="https://www.facebook.com/The.European.Experience.2016/?fref=ts">
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                                            <i class="zmdi zmdi-facebook btn waves-effect"></i>
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                                        </a>
 +
                                    </li>
 +
                                    <li>
 +
                                        <a href="https://twitter.com/igem_ionis">
 +
                                            <i class="zmdi zmdi-twitter btn waves-effect"></i>
 +
                                        </a>
 +
                                    </li>
  
 +
                                </ul>
 +
                                <a class="th_bt btn waves-effect waves-light" href="https://2016.igem.org/Team:Ionis_Paris/Events">Learn More
 +
                                    <i class="zmdi zmdi-long-arrow-right"></i>
 +
                                </a>
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
                <div class="col-xs-12 col-sm-6 fix_p_r">
 +
                    <div class="team_member_photo">
 +
                        <div class="team_slid">
 +
                            <div class="item waves-effect">
 +
                                <img src="https://static.igem.org/mediawiki/2016/6/63/European_experience.jpg" alt="">
 +
                                <div class=""></div>
 +
                                <div class="memberPhoto_border bg2"></div>
 +
                                <div class="memberPhoto_border bg3"></div>
 +
                                <div class="memberPhoto_border bg4"></div>
 +
                            </div>
 +
                            <div class="item waves-effect">
 +
                                <img src="https://static.igem.org/mediawiki/2016/1/18/IONIS_IGEM_EE2016_cocktail.png" alt="">
 +
                                <div class="memberPhoto_border bg1"></div>
 +
                                <div class="memberPhoto_border bg2"></div>
 +
                                <div class="memberPhoto_border bg3"></div>
 +
                                <div class="memberPhoto_border bg4"></div>
 +
                            </div>
 +
                            <div class="item waves-effect">
 +
                                <img src="https://static.igem.org/mediawiki/2016/b/b0/IONIS_IGEM_EE2016_home_group.png" alt="">
 +
                                <div class="memberPhoto_border bg1"></div>
 +
                                <div class="memberPhoto_border bg2"></div>
 +
                                <div class="memberPhoto_border bg3"></div>
 +
                                <div class="memberPhoto_border bg4"></div>
 +
                            </div>
 +
                        </div>
 +
                        <div class="teamslide_nav">
 +
                            <i class="zmdi zmdi-long-arrow-left testi_prev"></i>
 +
                            <i class="zmdi zmdi-long-arrow-right testi_next"></i>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
        </div>
 +
    </section>
 +
    <!-- ====START OUR TEAM AREA==== -->
  
−
         <h3><font color = "A2233C"> Protocol and tested concentrations </font></h3>
+
    <!-- ====START SERVICE AREA==== -->
 +
    <section class="service_area section-padding">
 +
         <div class="container">
 +
            <div class="row">
 +
                <div class="col-xs-12">
 +
                    <div class="section_title mb80">
 +
                        <h2 class="secHd text-center" style="color: #429fd7;">A multidisciplinary team</h2><br />
 +
                        <h5 class="smallHd text-center" style="color: #429fd7;">The iGEM IONIS team is developping its
 +
                            project through different axes. We are focused on two areas : <br /><br />the development of
 +
                            our prototype and the realisation of our own european jamboree, the European Experience.
 +
                        </h5>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
            <div class="row">
 +
                <div class="col-xs-12 col-sm-6 col-md-4">
 +
                    <div class="single_service text-center">
  
−
    <p> The protocol 12 (available <a href="https://2016.igem.org/Team:Ionis_Paris/Protocol_12"> <font color="DeepPink"> here </font></a>) was used to asses cell survival in presence of toluene. We tested a large range of concentrations (from 5ng/L to 5mg/L) to evaluate the impact of toluene on the <i> E.coli </i> DH5α cell growth. 5ng/L is a concentration closed to environmental toluene concentration.<br/>  
+
                        <h3>Biotechnology</h3>
−
An overnight culture of bacteria tranformed with our biosensor was realize. 300µL of this overnight culture was inoculated into  150mL of LB containing chloramphenicol at 25ng/mL. This culture was homogenize and separate into several falcon tubes. Tolune was then added to reach its desired concentration. Three replicates of each concentration were done to ensure statistical results.<br/>
+
                        <img src="https://static.igem.org/mediawiki/2016/0/01/IONIS_IGEM_supbiotech_shcool.png" alt="" />
−
The OD600 was then checked every hours during 8 hours.<br/>
+
                    </div>
−
</p>
+
                </div>
 +
                <div class="col-xs-12 col-sm-6 col-md-4">
 +
                    <div class="single_service text-center">
  
 +
                        <h3>Computer Science</h3>
 +
                        <img src="https://static.igem.org/mediawiki/2016/3/30/IONIS_IGEM_epita_shcool.png" alt="" />
  
−
            <h3><font color = "A2233C"> Results </font></h3>
 
  
−
    <p>The graph 1 shows the evolution of sample absorbance according to the time and the toluene concentration added in cell culture medium. The curve shows normal growth profile with a lag phase of three hours and then an exponential phase for all samples whether the toluene concentration added in the cell culture medium. <br/>
+
                    </div>
−
The toluene did not show a significant effect on cell growth at concentration of 5ng/L and 500ng/L. The growth curve shows the same profile than cells grew without toluene. At a concentration of 5mg/L of toluene in the medium, a small effect can be noticed. Four hours after toluene addition, the OD600 of cell grown in 5mg/L of toluene increased slowlier than the OD600 of cell grown without toluene.<br/>
+
                </div>
−
According to those results, bioluminescence test were realized with a toluene concentration comprised between 0 and 10 mg/L.
+
                <div class="col-xs-12 col-sm-6 col-md-4">
−
</p>
+
                    <div class="single_service text-center">
 +
                        <h3>Development</h3>
 +
                        <img src="https://static.igem.org/mediawiki/2016/0/04/IONIS_IGEM_epitech_shcool.png" alt="" />
 +
                    </div>
 +
                </div>
 +
                <div class="col-xs-12 col-sm-6 col-md-4">
 +
                    <div class="single_service text-center">
  
−
<br/>
+
                        <h3>Graphic Design</h3>
−
<br/>
+
                        <img src="https://static.igem.org/mediawiki/2016/5/5d/IONIS_IGEM_eartsup_shcool.png" alt="" />
−
<br/>
+
                    </div>
 +
                </div>
 +
                <div class="col-xs-12 col-sm-6 col-md-4">
 +
                    <div class="single_service text-center">
  
 +
                        <h3>Marketing & Communication</h3>
 +
                        <img src="https://static.igem.org/mediawiki/2016/6/69/IONIS_IGEM_ISTM_shcool.png" alt="" />
 +
                    </div>
 +
                </div>
 +
                <div class="col-xs-12 col-sm-6 col-md-4">
 +
                    <div class="single_service text-center">
  
 +
                        <h3>Aeronautics</h3>
 +
                        <img src="https://static.igem.org/mediawiki/2016/a/a5/IONIS_IGEM_IPSA_shcool.png" alt="" />
  
−
        <figure>
+
                    </div>
−
            <center><img src="https://static.igem.org/mediawiki/2016/1/1b/T--Ionis_Paris--Cell_survival_and_.png" alt=""></center>
+
                </div>
−
             <figcaption><p><i> Figure 1 : Evolution of E.coli DH5α grotwh depending on the toluene concentration added in the cell culture medium </i></p></figcaption>
+
             </div>
−
        </figure>
+
        </div>
 +
    </section>
 +
    <!-- ====END SERVICE AREA==== -->
  
  
−
                          <h2 class="secHd"> Biosensor characterization </h2>
+
 
  
 +
 +
    <!-- ====START PARTNER==== -->
 +
    <div class="partner_area">
 +
        <div class="container">
 +
            <div class="row">
 +
                <div class="col-xs-12">
 +
                    <div class="section_title mb80">
 +
                        <h5 class="smallHd text-center">Our</h5>
 +
                        <h2 class="secHd text-center">SPONSORS AND PARTNERS </h2>
 +
                    </div>
 +
                </div>
 +
                <div class="col-md-12">
 +
                    <div class="partner_slider">
 +
                      <div class="item">
 +
                            <img src="https://static.igem.org/mediawiki/2016/0/0e/IONIS_IGEM_partener_ARPF.png" alt="partner slider" />
 +
                        </div>
 +
                      <div class="item">
 +
                            <img src="https://static.igem.org/mediawiki/2016/0/08/IONIS_IGEM_partener_bouygues.png" alt="partner slider" />
 +
                        </div>
 +
                      <div class="item">
 +
                            <img src="https://static.igem.org/mediawiki/2016/3/36/IONIS_IGEM_partener_Bio_new_england.png" alt="partner slider" />
 +
                        </div>
  
−
 
−
                    <h3><font color = "A2233C"> Protocol and tested concentrations </font> </h3>
 
−
 
−
    <p>We used the protocol 13 (available <a href="https://2016.igem.org/Team:Ionis_Paris/Protocol_13"> <font color="DeepPink"> here </font> </a>)and decided to test several concentration of toluene (0ng/L, 10ng/L, 100ng/L, 10µg/L and 10mg/L).Bioluminescence assays were realized several times after toluene addition (1h, 3h, 4h30 and 5h30). <br/>
 
−
Bacteria transformed with our biosensor used in each assays come from the same culture (OD = 0.1). This culture was well mixed and divided in 50 mL falcon. Toluene was then added in each Falcon. <br/>
 
−
All the test were realized in triplicates. <br/>
 
−
 
−
Bioluminescence assay were realized quickly after substrate addition and the bioluminescence intensity was measured using <a href="https://www.berthold.com/en/bio/monochromator_multilabel_microplate_reader_Mithras2_LB943"> <font color="DeepPink"> Mithras² LB 943 Monochromator Multimode Reader</font></a>.This machine was kindly lend by Berthold company for 3 days in order to let us realized the bioluminescence measurement. <br/>
 
−
Sample's ODs were also measured. <br/>
 
−
 
−
Several negative control were realized :  <br/>
 
−
</p>
 
−
 
−
    <li> <p>Measurement of LB bioluminescence intensity to determine the background noise</p></li>
 
−
    <li><p>Measurement of LB + toluene bioluminescence intensity (effect of toluene on the bioluminescent intensity)</p></li>
 
−
    <li><p>Measurement the bioluminescence intensity of bacteria transformed with a genetic construction that do not contain the gaussia luciferase gene (Pr-XylR).</p></li>
 
−
 
−
  <p>A positive control using Pr-Gluc (BBG1) to test our substrate efficiency and the luminometer parameters could have been made during the three days of measurement. However, this biobrick was created afterwards. This biobrick will serve as a future control for all bioluminescence measurement in iGEM project to ensure the substrate efficiency.
 
−
</p>
 
−
 
−
          <h3><font color = "A2233C"> Preliminary considerations </font> </h3>
 
−
<br/>
 
−
 
−
      <b><font color = "D32C50"> Toluene does not have auto-bioluminescence </font> </b>
 
−
 
−
  <p>We determine the effect of adding toluene to cell culture medium on bioluminescence intensity. As shown on the graph below the bioluminescence intensity of the LB + toluene at 10 mg/L (47.71 RLU) do not significantly differ from the bioluminescence intensity of the LB (48.93 RLU). The little bioluminescence found was due to the substrate added in our sample. <br/>
 
−
Therefore, toluene addition in our sample will not impact their bioluminescence intensity.</p>
 
−
 
−
 
−
<div class="container">
 
−
<div class="row">
 
−
 
−
 
−
  <div class="col-sm-5">
 
−
    <center><table>
 
−
                <tr>
 
−
                  <td><p>  </p></td>
 
−
                  <td><p>LB without toluene</p></td>
 
−
                  <td><p>LB with toluene at 10mg/L</p></td>
 
−
                </tr>
 
−
 
−
                <tr>
 
−
                  <td><p>Mean</p></td>
 
−
                  <td><p>48.93 RLU</p></td>
 
−
                  <td><p>47.71 RLU</p></td>
 
−
                </tr>
 
−
 
−
                <tr>
 
−
                  <td><p>Standard deviation (SD)</p></td>
 
−
                  <td><p>6.96</p></td>
 
−
                  <td><p>9.55</p></td>
 
−
                </tr>
 
−
 
−
                <tr>
 
−
                  <td><p>Std. error of mean(SEM)</p></td>
 
−
                  <td><p>1.87</p></td>
 
−
                  <td><p>2.55</p></td>
 
−
                </tr>
 
−
 
−
              </table></center>
 
−
      </div>
 
−
 
−
      <div class="col-sm-5">
 
−
        <figure>
 
−
              <center><img src="https://static.igem.org/mediawiki/2016/4/43/T--Ionis_Paris--Toluene_bioluminescence_and_.jpg" alt=""></center>
 
−
              <figcaption><p><i> Figure 2 : Bioluminescence intensity expressed in RLU of LB and LB with toluene at 10mg/L. Mann whitney statistical test was performed to asses the effect of toluene on LB auto-bioluminescence (ns: non significant). </i></p></figcaption>
 
−
        </figure>
 
−
 
−
      </div>
 
−
 
−
</div>
 
−
</div>
 
−
 
−
 
−
<br/>
 
−
 
−
      <b><font color = "D32C50"> Bacteria E.coli have little auto-bioluminescence </font> </b>
 
−
 
−
    <p> Bacteria transformed with a genetic construction that does not contain Gaussia luciferase gene were used in this study. The auto-bioluminescence of E.coli cells was assessed by comparate <i>E.coli</i> bacteria bioluminescence intensity to LB bioluminescence intensity. As shown on the figure 3, the bioluminescence intensity of <i>E.coli </i> cells (53.14 RLU) was a little superior to LB bioluminescence intensity (48.93 RLU) but not significantly superior. <i>E.coli</i> cells have little auto-bioluminescence. However, this bioluminescence in greatly inferior to the one produce by our biosensor in presence of pollutant (results presented after) <p>
 
−
 
−
 
−
 
−
<div class="container">
 
−
<div class="row">
 
−
 
−
<div class="col-sm-5">
 
−
 
−
      <center><table>
 
−
                <tr>
 
−
                  <td><p>  </p></td>
 
−
                  <td><p>LB without toluene</p></td>
 
−
                  <td><p>BB12 without toluene</p></td>
 
−
                </tr>
 
−
 
−
                <tr>
 
−
                  <td><p>Mean</p></td>
 
−
                  <td><p>48.93 RLU</p></td>
 
−
                  <td><p>53.14 RLU</p></td>
 
−
                </tr>
 
−
 
−
                <tr>
 
−
                  <td><p>Standard deviation (SD)</p></td>
 
−
                  <td><p>6.96</p></td>
 
−
                  <td><p>11.34</p></td>
 
−
                </tr>
 
−
 
−
                <tr>
 
−
                  <td><p>Std. error of mean(SEM)</p></td>
 
−
                  <td><p>1.86</p></td>
 
−
                  <td><p>3.03</p></td>
 
−
                </tr>
 
−
 
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              <figcaption><p><i> Figure 3 : Bioluminescence intensity expressed in RLU of LB and LB + <i>E.coli </i> cells. Mann whitney statistical test was performed (ns: non significant).</i></p></figcaption>
 
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            <b><font color = "D32C50"> Little leakage in Gaussia luciferase expression</font> </b>
 
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−
  <p> We investigate the inductility of the Pu promoter to ensure that the gaussia luciferase is not constitutively produce in our biosensor. To do so we compared the bioluminescence intensity of bacteria containing our biosensor to the bioluminescence intensity of bacteria transformed with a genetic construction that do not contain the Gaussia luciferase gene (BB12 : Pr-RBS-XylR). <br/>
 
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−
The obtained results (figure 4) indicate that the gaussia luciferase gene is not produce in a constitutive manner in the cells. The bioluminescence intensity of our biosensor without toluene injection (158.86 RLU) is a little higher than the bioluminescence intensity of cells transformed with BB12 (53.14 RLU). However this bioluminescence intensity is significantly lower than the bioluminescence intensity of the cells transformed with our biosensor in presence of toluene (results presented in the next section)
 
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                      <td><p>  </p></td>
 
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                      <td><p>BB12 without toluene</p></td>
 
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                      <td><p>BB123 without toluene</p></td>
 
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                      <td><p>Mean</p></td>
 
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                      <td><p>53.14 RLU</p></td>
 
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                      <td><p>158.86 RLU</p></td>
 
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                      <td><p>Standard deviation (SD)</p></td>
 
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                      <td><p>11.34</p></td>
 
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                      <td><p>20.08</p></td>
 
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                      <td><p>Std. error of mean(SEM)</p></td>
 
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                      <td><p>3.03</p></td>
 
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                      <td><p>3.10</p></td>
 
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                  <center><img src="https://static.igem.org/mediawiki/2016/9/9f/T--Ionis_Paris--gaussia_and_.jpg" alt=""></center>
 
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                  <figcaption><p><i> Figure 4 : Bioluminescence intensity expressed in RLU of <i>E.coli </i> cells transformed with a construction genetic that contain (BB123) or do not contain (BB12) the gaussia gene. A little leakage of the gaussia gene can be seen as the bioluminescence intensity of the sample that contain bacteria transformed with our biosensor is significantly superior to the one that contain bacteria transformed with Pr-RBS-XylR. Mann whitney statistical test was performed (*** : extremely significant).</i></p></figcaption>
 
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<h3><font color = "A2233C"> Data processing </font></h3>
 
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<p> Due to the little LB background noise and leakage in gaussia luciferase synthesis, obtain bioluminescence intensity results were treated as followed. The background noise (LB+toluene) was subtracted to each sample’s bioluminescence intensity. Those latest were then expressed depending on the negative control (bacteria transformed with our biosensor in a medium without toluene, C0).</p>
 
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<h3><font color = "A2233C"> Results </font> </h3>
 
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<b><font color = "D32C50"> Investigation of the best measurement time</font> </b>
 
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  <p>It was first necessary to determine when, after toluene injection, bioluminescence results were the most relevant. To do so, we determine the bioluminescence intensity of our sample (bacteria transformed with our biosensor within LB medium containing different toluene concentration) several times after toluene addition. <br/>
 
−
The bioluminescence intensity of the negative control was also determine at each time after toluene addition and bioluminescent results for each assay were normalized on the corresponding negative control bioluminescence intensity. This normalization was necessary as we are dealing with several incubation time. This enable us to handle the bacteria OD increase within our samples and ensure that an increase in sample’s bioluminescence intensity is not correlated with an increase in cells concentration.<br/>
 
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−
The graphs presented below represent the bioluminescence intensity of the sample depending on the time after a toluene addition for a fix concentration of toluene injected.
 
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                <figcaption><p><i> Figure 5 : Evolution of bioluminescence intensity at different times after toluene addition (10ng/L). The bioluminescence intensity of each sample is normalized on the bioluminescence intensity of the corresponding negative control. Mann whitney statistical test was performed (ns : non significant, * : significant, ** : very significant, *** : extremely significant) </i></p></figcapion>
 
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                <figcaption><p><i> Figure 6 : Evolution of bioluminescence intensity at different times after toluene addition (100ng/L). The bioluminescence intensity of each sample is normalized on the bioluminescence intensity of the corresponding negative control. Mann whitney statistical test was performed (ns : non significant, * : significant, ** : very significant, *** : extremely significant)</i></p></figcaption>
 
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<figcaption><p><i> Figure 7 : Evolution of bioluminescence intensity at different times after toluene addition (10µg/L). The bioluminescence intensity of each sample is normalized on the bioluminescence intensity of the corresponding negative control. Mann whitney statistical test was performed (ns : non significant, * : significant, ** : very significant, *** : extremely significant)</i></p></figcaption>
 
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              <figcaption><p><i> Figure 8 : Evolution of bioluminescence intensity at different times after toluene addition (10mg/L). The bioluminescence intensity of each sample is normalized on the bioluminescence intensity of the corresponding negative control. Mann whitney statistical test was performed (ns : non significant, * : significant, ** : very significant, *** : extremely significant)</i></p></figcaption>
 
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  <p>The bioluminescence intensity of all our samples was compared to the bioluminescence intensity of the negative control for each time after toluene injection. Mann Whitney statistics test were realized to determine whether the bioluminescence intensity of the sample was significantly higher than the bioluminescence intensity of the negative control.<br/>
 
−
On the graphics we can notice that the bioluminescence intensity increase as the time after toluene injection increase. However, for the highest concentration (10 mg/L), a decrease in the bioluminescence intensity can be notice 5h30 after toluene injection. It is due to the accumulation of gaussia luciferase and the impact of toluene on the cell metabolism and especially protein synthesis (for more information click <a href = "https://2016.igem.org/Team:Ionis_Paris/E.coli"> <font color="DeepPink"> here </font></a>). <br/>
 
−
Therefore, it is essential to realize our bioluminescence test at a precise time after sampling. As we want to detect environmental toluene concentration (less than 20ng/L), it is to be suitable to wait 5h30 before proceeding to the bioluminescence test.
 
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        <b><font color = "D32C50"> Investigation of detectable concentration 5h30 after toluene addition to the cells medium</font> </b>
 
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        <p>As we determined that it was preferable to wait 5h30 before realizing the bioluminescence assay, we decided to focus on the results obtain for this time after toluene addition in the cells medium. We studied the evolution of the bioluminescence intensity according to the toluene concentration added in the cells medium 5h30 before. Those tests were realize in triplicates.<br/>
 
 
   
 
   
−
First of all, we wanted to ensure that the OD of the different samples is the same 5h30 after bacteria inoculation in presence of toluene. As shown on the graph below, the sample’s ODs are quite similar going from 1.464 to 1.61.  
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              <figcaption><p><i> Figure 9 : Sample's ODs depending on the toluene concentration added 5h30 before </i></p></figcaption>
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      <p>The bioluminescence intensity of each samples was then determined and shown depending on the toluene concentration. As we are dealing with extremely different toluene concentration, it was preferable to give our results in function of the logarithm of the toluene concentration. The bioluminescence intensity data in RLU for each replicates (R1, R2, R3) are given in the table below and represented on the graph 10.
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                            <td><p>Toluene concentration</p></td>
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                            <td><p>Logarithm of toluene concentration</p></td>
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                            <td><p>Mean - R1 (RLU)</p></td>
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                            <td><p>SD - R1</p></td>
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                            <td><p>Mean - R2 (RLU)</p></td>
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                            <td><p>SD - R2</p></td>
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                            <td><p>Mean - R3 (RLU)</p></td>
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                            <td><p>SD - R3</p></td>
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                            <td><p>0 ng/L</p></td>
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                            <td><p>0</p></td>
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                            <td><p>180.2</p></td>
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                            <td><p>16.0</p></td>
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                            <td><p>146.5</p></td>
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                            <td><p>23.4</p></td>
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                            <td><p>167.9</p></td>
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                            <td><p>11.8</p></td>
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                            <td><p>10 ng/L</p></td>
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                            <td><p>1</p></td>
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                            <td><p>1007.3</p></td>
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                            <td><p>210.2</p></td>
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                            <td><p>337.7</p></td>
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                            <td><p>39.3</p></td>
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                            <td><p>1041.9</p></td>
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                            <td><p>139.4</p></td>
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                            <td><p>100 ng/L</p></td>
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                            <td><p>2</p></td>
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                            <td><p>754.4</p></td>
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                            <td><p>150.4</p></td>
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                            <td><p>762.4</p></td>
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                            <td><p>77.9</p></td>
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                            <td><p>1823.1</p></td>
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                            <td><p>243.1</p></td>
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                            <td><p>10 µg/L</p></td>
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                            <td><p>4</p></td>
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                            <td><p>1110.4</p></td>
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                            <td><p>150.0</p></td>
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                            <td><p>1373.8</p></td>
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                            <td><p>162.4</p></td>
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                            <td><p>2073.7</p></td>
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                            <td><p>202.9</p></td>
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                            <td><p>10 mg/L</p></td>
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                            <td><p>7</p></td>
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                            <td><p>882.8</p></td>
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                            <td><p>131.6</p></td>
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                            <td><p>2485.4</p></td>
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                            <td><p>259.5</p></td>
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                            <td><p>1247.2</p></td>
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                            <td><p>151.4</p></td>
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        <figcaption><p><i> Figure 10 : Evolution of bioluminescence intensity for each replicate depending on the logarithm of the toluene concentration injected 5h30 before</i></p></figcaption>
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<p>As shown on the graph, the obtained data differ from one replicates to another due to difference in the metabolism which is normal when working with living organism. However the curve profile stay is the same : the bioluminescence intensity of the sample increases until a toluene concentration of 10 µg/,  then it reaches a plateau for two replicates and continues to increase for the other replicate.<br/>
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Those results were pooled in order to draw a standard curve and being able to predict toluene concentration in a sample according to the bioluminescence intensity.
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<b><font color = "D32C50"> Quantification of environmentally relevant toluene concentration </font> </b>
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  <p>The figure 11 shows the evolution of the bioluminescence intensity depending on the logarithm of toluene concentration. For a toluene concentration inferior to 10 µg/L (10,000 ng/L : log(4)) the curve increases steadily, then the curve reach a plateau. This can be due to gaussia accumulation in the cells. <br/>
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We were able to detect environmentally relevant toluene concentration and as the standard curve is linear for a toluene concentration inferior to 10µg/L, we are able to quantify the toluene concentration of a given sample.
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                          <td><p>0 ng/L</p></td>
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                          <td><p>10 ng/L</p></td>
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                          <td><p>100 ng/L</p></td>
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                          <td><p>10 µg/L</p></td>
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                          <td><p>10 mg/L</p></td>
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                        <tr>
+
−
                          <td><p>Mean (RLU)</p></td>
+
−
                          <td><p>164,84</p></td>
+
−
                          <td><p>795,63</p></td>
+
−
                          <td><p>1139,88</p></td>
+
−
                          <td><p>1519,28</p></td>
+
−
                          <td><p>1538,45</p></td>
+
−
                        </tr>
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−
+
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                          <td><p>Standard deviation</p></td>
+
−
                          <td><p>17,62</p></td>
+
−
                          <td><p>129,73</p></td>
+
−
                          <td><p>146,68</p></td>
+
−
                          <td><p>171,75</p></td>
+
−
                          <td><p>180,84</p></td>
+
−
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                          <td><p>Induction percentage</p></td>
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                          <td><p></p></td>
+
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                          <td><p>383%</p></td>
+
−
                          <td><p>592%</p></td>
+
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                          <td><p>822%</p></td>
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−
                          <td><p>833%</p></td>
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                      <figcaption><p><i> Figure 11 : Evolution of bioluminescence intensity for pooled of the replicate depending on the logarithm of the toluene concentration injected 5h30 before. Correlation between toluene concentration in the cell culture medium and the bioluminescence intensity </i></p></figcaption>
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<h3><font color = "A2233C"> Comparison with existing methods </font> </h3>
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<h3><font color = "A2233C"> Perspectives and improvement  </font> </h3>
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<p>Improve standard curve at low concentration <br/>
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Determine the lowest concentration detectable
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</p>
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Latest revision as of 01:06, 2 December 2016

What is

Quantifly ?

(VOC)

VOLATILE ORGANIC COMPOUNDS

Nowadays, Volatile Organic Compounds (VOCs) are commonly found pollutants that have been proven present in a wide variety of everyday life situations. Though they are dangerous and present almost everywhere, VOCs are not efficiently detected. Usual detection devices are very unprecise or require a very long exposure time.

Bacteria

Biosensor

This is why we developed the Quantifly project : a combination of a bacterial biosensor, perfectly adapted to on-field measurements, and an innovative drone that will allow us to easily and rapidly quantify the amount of VOCs in the air. The biosensor relies on bioluminescence, a biochemical phenomenon that is commonly used in cell imaging. We are trying to develop a new application for bioluminescence that should allow us to create a biosensor of a new kind, entirely different from the existing devices.

drone
Bacteria

Drone

Concerning the drone, it is designed to safely contain our organisms and carry them on the field, thus acting as a mobile detection platform.

Check out the Video