Difference between revisions of "Team:Ionis Paris"

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    <link href='https://fonts.googleapis.com/css?family=Merriweather:400,700,700italic,400italic,300italic,300'
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        <!-- Nos feuilles de style -->
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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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          rel='stylesheet' type='text/css'>
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        <!-- ====BREADCUM START==== -->  
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    <!-- Nos feuilles de style -->
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        <section class="blog_banner_ecoli">
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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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                         <div class="banner_title">
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    <!-- ====HEADER PARALUX==== -->
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                             <h1 id="back_to_the_top">Proof of Concept</h1>
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        <!-- ====BREADCUM END==== -->
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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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    <!-- ====FEATURE AERE==== -->
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    <!--  <section class="feature_area" id="services">
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                          <h4>Branding</h4>
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                          <div class="single_cap">
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                          <p>As a business owner or manager, the decision to opt faor offshore software can be difficult.</p>
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                          <a class="th_bt btn waves-effect" href="#!">Read More</a>
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                          <i class="zmdi zmdi-globe-alt"></i>
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                          <h4>Web Development</h4>
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                          <div class="single_cap">
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                          <p>As a bu siness owner or manager, the decision to opt faor offshore software can be difficult.</p>
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                          <a class="th_bt btn waves-effect" href="#!">Read More</a>
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                          <i class="zmdi zmdi-apple"></i>
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                          <h4>Mobile Application</h4>
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                          <p>As a bu siness owner or manager, the decision to opt faor offshore software can be difficult.</p>
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                          <a class="th_bt btn waves-effect" href="#!">Read More</a>
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                          <h4>Photography</h4>
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                          <p>As a bu siness owner or manager, the decision to opt faor offshore software can be difficult.</p>
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                          <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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      </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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                        <h2 class="sec text-center">WHAT IS</h2>
 +
                        <h2 class="secHd text-center ">Quantifly ?</h2>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
            <div class="col-xs-12 col-sm-6">
 +
                <div class="about_left">
 +
                    <img src="https://static.igem.org/mediawiki/2016/2/23/Ville.png" alt="" />
 +
                </div>
 +
            </div>
 +
            <div class="col-xs-12 col-sm-6">
 +
                <div class="section_title">
 +
                    <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 proved
 +
                        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 requires a very long exposure time.</p>
  
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              <h3><font color = "A2233C">Protocol</font> </h3>
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                </div>
 +
            </div>
 +
        </div>
 +
        </div>
 +
    </section>
 +
    <!-- ====ABOUT US==== -->
 +
    <!-- ====ABOUT US==== -->
 +
    <section id="about" class="about_area section-padding">
 +
        <div class="container">
 +
            <div class="row">
 +
                <div class="col-xs-12 col-sm-6">
 +
                    <div class="section_title">
 +
                        <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>
 +
                        <p class="ab_text">If you’ve followed along and done the steps above, you’ve created a good
 +
                            starting place. You’re going to need to make sure your pages are optimized properly.</p>
 +
                    </div>
 +
                </div>
 +
                <div class="col-xs-12 col-sm-5">
  
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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/>
 
−
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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              <h3><font color = "A2233C">Results</font></h3>
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                </div>
 +
                <div class="about_left">
 +
                    <img src="https://static.igem.org/mediawiki/2016/7/7d/Biosensor.png" alt="" />
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                </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>
−
            </tr>
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    <!-- ====ABOUT US==== -->
 +
    <!-- ====ABOUT US==== -->
 +
    <section id="about" class="about_area section-padding">
 +
        <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">
 +
                    <div class="section_title">
 +
                        <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
 +
                            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>
+
             </div>
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             </tr>
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        </div>
 +
    </section>
 +
    <!-- ====ABOUT US==== -->
 +
    <!-- ====START SKILL AND VEDIO TABLE==== -->
 +
    <section class="skill_and_video">
 +
        <div class="video_area">
 +
            <div class="video_control">
 +
                <div class="dtable">
 +
                    <div class="dcell">
 +
                        <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>
 +
                        <h4>Check out the Video</h4>
 +
                    </div>
 +
                </div>
 +
             </div>
 +
        </div>
  
−
            <tr>
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        <div class="row">
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              <td><p> 2h</p>  </td>
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             <div class="col-md-3 col-xs-6">
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              <td><p> Weight</p>  </td>
+
−
             </tr>
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−
             <tr>
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             </div>
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              <td><p> 2h30</p>  </td>
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        </div>
−
              <td><p> Weight</p> </td>
+
−
            </tr>
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−
          </table></center>
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        </div>
 +
        </div>
 +
        </div>
 +
        </div>
 +
    </section>
 +
    <!-- ====END SKILL AND VEDIO TABLE==== -->
  
−
  <p> As shown in the table above, after 6 hours the weight decrease …….  compared to the starting weight. <br/>
 
−
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/>
 
−
The stock solution of toluene will be prepare again for each replicates.
 
−
</p>
 
  
 +
    <!-- ====START OUR TEAM AREA==== -->
 +
    <section class="our_team">
 +
        <div class="container">
 +
            <div class="row">
 +
                <div class="col-xs-12">
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                    <div class="section_title mb80">
 +
                        <h5 class="smallHd text-center">OUR BEST INTER-TEAM
 +
                        </h5>
 +
                        <h2 class="secHd text-center">COLLABORATION</h2>
 +
                    </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">
  
 +
                            <div class="item">
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                                <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 weekend, by coming to Paris and being able to live the “science dream” in the city of light !
 +
                                </p>
 +
                                <ul class="team_social">
 +
                                    <li>
 +
                                        <a href="#!">
 +
                                            <i class="zmdi zmdi-facebook btn waves-effect"></i>
 +
                                        </a>
 +
                                    </li>
 +
                                    <li>
 +
                                        <a href="#!">
 +
                                            <i class="zmdi zmdi-twitter btn waves-effect"></i>
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                                        </a>
 +
                                    </li>
 +
                                    <li>
 +
                                        <a href="#!">
 +
                                            <i class="zmdi zmdi-dribbble btn waves-effect"></i>
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                                        </a>
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                                    </li>
 +
                                    <li>
 +
                                        <a href="#!">
 +
                                            <i class="zmdi zmdi-behance btn waves-effect"></i>
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                                        </a>
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                                    </li>
 +
                                    <li>
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                                        <a href="#!">
 +
                                            <i class="zmdi zmdi-pinterest btn waves-effect"></i>
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                                        </a>
 +
                                    </li>
 +
                                </ul>
 +
                                <a class="th_bt btn waves-effect waves-light" href="#!">Learn More
 +
                                    <i class="zmdi zmdi-long-arrow-right"></i>
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                                </a>
 +
                            </div>
 +
                            <div class="item">
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                                <h1>Timothy<br>Dean</h1>
 +
                                <h6>Creative Director</h6>
 +
                                <p>If you’ve followed along and done the steps above, you’ve created  good starting place. You’re going to need to make sure your pages are optimized properly. This will ensure you get your page ranked high in the search engines.</p>
 +
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                                <h1>Ralph <br>Ramirez</h1>
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                                <h6>Creative Director</h6>
 +
                                <p>If you’ve followed along and done the steps above, you’ve created  good starting place. You’re going to need to make sure your pages are optimized properly. This will ensure you get your page ranked high in the search engines.</p>
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                                <a class="th_bt btn waves-effect waves-light" href="#!">Learn More
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 +
        </div>
 +
    </section>
 +
    <!-- ====START OUR TEAM AREA==== -->
 +
    <!-- ====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">
  
−
                                    <h2 class="secHd"> Survival test to toluene </h2>
+
                        <h3>Biotechnology</h3>
 +
                        <img src="https://static.igem.org/mediawiki/2016/0/01/IONIS_IGEM_supbiotech_shcool.png" alt="" />
 +
                    </div>
 +
                </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"> Protocol and tested concentrations </font></h3>
 
  
−
    <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/>  
+
                    </div>
−
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/>
+
                </div>
−
The OD600 was then checked every hours during 8 hours.<br/>
+
                <div class="col-xs-12 col-sm-6 col-md-4">
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</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">
  
 +
                        <h3>Graphic Design</h3>
 +
                        <img src="https://static.igem.org/mediawiki/2016/5/5d/IONIS_IGEM_eartsup_shcool.png" alt="" />
 +
                    </div>
 +
                </div>
 +
                <div class="col-xs-12 col-sm-6 col-md-4">
 +
                    <div class="single_service text-center">
  
−
            <h3><font color = "A2233C"> Results </font></h3>
+
                        <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">
  
−
    <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/>
+
                        <h3>Aeronautics</h3>
−
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/>
+
                        <img src="https://static.igem.org/mediawiki/2016/a/a5/IONIS_IGEM_IPSA_shcool.png" alt="" />
−
According to those results, bioluminescence test were realized with a toluene concentration comprised between 0 and 10 mg/L.
+
−
</p>
+
  
−
<br/>
+
                    </div>
−
<br/>
+
                </div>
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<br/>
+
            </div>
 +
        </div>
 +
    </section>
 +
    <!-- ====END SERVICE AREA==== -->
  
  
 +
    <!-- ====START WHY CHOOSE==== -->
 +
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 +
        <div class="container">
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                    <div class="section_title mb80">
 +
                        <h2 class="secHd text-center" style="color: #e62562;">What we aim to do</h2><br />
 +
                        <h5 class="smallHd text-center" style="color: #e62562;">Though our project aims to realise a lot
 +
                            in a short period of time, we have objectives<br /><br /> that would develop further our
 +
                            project Quantifly :</h5>
 +
                    </div>
 +
                </div>
 +
            </div>
 +
            <div class="row checkBGFull">
 +
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 +
                        </div>
 +
                        <div class="checkText">
 +
                            <h4>cost effectiveness</h4>
 +
                            <p>Also, we aim to develop metabolic pathways in order to add a degradation ability to the
 +
                                cell in complement of detecting and measuring pollutant levels in the air.</p>
 +
                        </div>
 +
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 +
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                        </div>
 +
                        <div class="checkText">
 +
                            <h4>unique ideas</h4>
 +
                            <p>Last but not least, we want to popularize even more synthetic biology by participating in
 +
                                other science conferences during all the year 2016.</p>
 +
                        </div>
 +
                    </div>
 +
                </div>
  
−
        <figure>
+
            </div>
−
             <center><img src="https://static.igem.org/mediawiki/2016/1/1b/T--Ionis_Paris--Cell_survival_and_.png" alt=""></center>
+
             <div class="row checkBGFull">
−
            <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 class="col-xs-12 col-sm-6 ourFeaturesContent section-padding">
−
        </figure>
+
                    <div class="single_checkCont">
 +
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 +
                            <i class="zmdi zmdi-account-add"></i>
 +
                        </div>
 +
                        <div class="checkText">
 +
                            <h4>reliability</h4>
 +
                            <p>We wish to compare our measurement results with already existing results gathered with
 +
                                different methods, in order to evaluate our biosensor efficiency.</p>
 +
                        </div>
 +
                    </div>
 +
                    <div class="single_checkCont">
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                        </div>
 +
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 +
                            <h4>customer relationship</h4>
 +
                            <p>We want to optimize our bacteria by
 +
                                enhancing the bioluminescence signal emitted by the cell.</p>
 +
                        </div>
 +
                    </div>
  
 +
                </div>
  
−
                          <h2 class="secHd"> Biosensor characterization </h2>
+
            </div>
 +
        </div>
 +
    </section>
 +
    <!-- ====END WHY CHOOSE==== -->
  
  
 +
    <!-- ====START PROCESS TABLE==== -->
 +
    <section class="process_area section-padding">
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                    <div class="section_title mb80">
 +
                        <h5 class="smallHd text-center">the simple step of</h5>
 +
                        <h2 class="secHd text-center">our working process</h2>
 +
                    </div>
 +
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 +
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 +
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 +
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                                <p>RSS, (Really Simple Syndication) has become so popular today and is being referred to
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                                <p>RSS, (Really Simple Syndication) has become so popular today and is being referred to
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    </section>
 +
    <!-- ====END PROCESS TABLE==== -->
  
−
                    <h3><font color = "A2233C"> Protocol and tested concentrations </font> </h3>
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                        <h5 class="smallHd text-center">Our</h5>
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                        <h2 class="secHd text-center">SPONSORS AND PARTENAIRS </h2>
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    <!-- ====END PARTNER==== -->
  
−
    <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/>
+
    <!-- ====END BLOG TABLE==== -->
−
Sample's ODs were also measured. <br/>
+
  
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Several negative control were realized :  <br/>
 
−
</p>
 
  
−
    <li> <p>Measurement of LB bioluminescence intensity to determine the background noise</p></li>
+
    <!-- ====START SOCIAL Link==== -->
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    <li><p>Measurement of LB + toluene bioluminescence intensity (effect of toluene on the bioluminescent intensity)</p></li>
+
    <div class="footer_social">
−
    <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>
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−
  <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.
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</p>
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            </div>
 +
        </div>
 +
    </div>
 +
    <!-- ====END FOOTER TOP==== -->
  
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          <h3><font color = "A2233C"> Preliminary considerations </font> </h3>
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     <!-- ====START FOOTER AREA==== -->
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<br/>
+
    <footer class="footer_area">
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         <div class="footer_middle">
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      <b><font color = "D32C50"> Toluene does not have auto-bioluminescence </font> </b>
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             <div class="container">
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+
−
  <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>
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<div class="container">
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<div class="row">
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  <div class="col-sm-5">
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    <center><table>
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                <tr>
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                  <td><p>  </p></td>
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−
                  <td><p>LB without toluene</p></td>
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                  <td><p>LB with toluene at 10mg/L</p></td>
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                </tr>
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                <tr>
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                  <td><p>Mean</p></td>
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                  <td><p>48.93 RLU</p></td>
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−
                  <td><p>47.71 RLU</p></td>
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−
                </tr>
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−
                <tr>
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−
                  <td><p>Standard deviation (SD)</p></td>
+
−
                  <td><p>6.96</p></td>
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−
                  <td><p>9.55</p></td>
+
−
                </tr>
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+
−
                <tr>
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                  <td><p>Std. error of mean(SEM)</p></td>
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−
                  <td><p>1.87</p></td>
+
−
                  <td><p>2.55</p></td>
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                </tr>
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              </table></center>
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−
      </div>
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      <div class="col-sm-5">
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        <figure>
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              <center><img src="https://static.igem.org/mediawiki/2016/4/43/T--Ionis_Paris--Toluene_bioluminescence_and_.jpg" alt=""></center>
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−
              <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>
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        </figure>
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      </div>
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</div>
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</div>
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<br/>
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      <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>
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<div class="container">
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<div class="row">
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      <center><table>
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                <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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+
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              </table></center>
+
−
 
+
−
</div>
+
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+
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<div class="col-sm-5">
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−
    <figure>
+
−
              <center><img src="https://static.igem.org/mediawiki/2016/6/6c/T--Ionis_Paris--bacteria_bioluminescence_and_.jpg" alt=""></center>
+
−
              <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>
+
−
      </figure>
+
−
 
+
−
</div>
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+
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</div>
+
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</div>
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+
−
<br/>
+
−
 
+
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            <b><font color = "D32C50"> Little leakage in Gaussia luciferase expression</font> </b>
+
−
 
+
−
  <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/>
+
−
 
+
−
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)
+
−
<p>
+
−
 
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−
 
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<div class="container">
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−
<div class="row">
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<div class="col-sm-5">
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        <center><table>
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−
                    <tr>
+
−
                      <td><p>  </p></td>
+
−
                      <td><p>BB12 without toluene</p></td>
+
−
                      <td><p>BB123 without toluene</p></td>
+
−
                    </tr>
+
−
 
+
−
                    <tr>
+
−
                      <td><p>Mean</p></td>
+
−
                      <td><p>53.14 RLU</p></td>
+
−
                      <td><p>158.86 RLU</p></td>
+
−
                    </tr>
+
−
 
+
−
                    <tr>
+
−
                      <td><p>Standard deviation (SD)</p></td>
+
−
                      <td><p>11.34</p></td>
+
−
                      <td><p>20.08</p></td>
+
−
                    </tr>
+
−
 
+
−
                    <tr>
+
−
                      <td><p>Std. error of mean(SEM)</p></td>
+
−
                      <td><p>3.03</p></td>
+
−
                      <td><p>3.10</p></td>
+
−
                    </tr>
+
−
 
+
−
              </table></center>
+
−
 
+
−
</div>
+
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+
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+
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<div class="col-sm-5">
+
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+
−
        <figure>
+
−
                  <center><img src="https://static.igem.org/mediawiki/2016/9/9f/T--Ionis_Paris--gaussia_and_.jpg" alt=""></center>
+
−
                  <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>
+
−
         </figure>
+
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+
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+
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</div>
+
−
</div>
+
−
</div>
+
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+
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+
−
<h3><font color = "A2233C"> Data processing </font></h3>
+
−
 
+
−
<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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+
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+
−
<h3><font color = "A2233C"> Results </font> </h3>
+
−
 
+
−
<b><font color = "D32C50"> Investigation of the best measurement time</font> </b>
+
−
 
+
−
 
+
−
  <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/>
+
−
 
+
−
 
+
−
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.
+
−
</p>
+
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        <figure>
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                <center><img src="https://static.igem.org/mediawiki/2016/b/bd/T--Ionis_Paris--10ng_time_and_.png" alt=""></center>
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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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                <center><img src="https://static.igem.org/mediawiki/2016/d/d8/T--Ionis_Paris--100ng_time_and_.png" alt=""></center>
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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/>
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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/>
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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/>
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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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              <center><img src="https://static.igem.org/mediawiki/2016/7/76/T--Ionis_Paris--OD_and_.png" alt=""></center>
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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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        </figure>
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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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              <center><table>
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                          <tr>
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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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                          </tr>
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                          <tr>
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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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                          </tr>
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                          <tr>
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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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        <center><img src="https://static.igem.org/mediawiki/2016/3/37/T--Ionis_Paris--T4_nonpool_and_.png" alt=""></center>
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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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<br/>
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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></p></td>
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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>
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                        <tr>
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                          <td><p>Mean (RLU)</p></td>
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−
                          <td><p>164,84</p></td>
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                          <td><p>795,63</p></td>
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                          <td><p>1139,88</p></td>
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                          <td><p>1519,28</p></td>
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                          <td><p>1538,45</p></td>
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                        </tr>
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                        <tr>
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                          <td><p>Standard deviation</p></td>
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−
                          <td><p>17,62</p></td>
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−
                          <td><p>129,73</p></td>
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                          <td><p>146,68</p></td>
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                          <td><p>171,75</p></td>
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                          <td><p>180,84</p></td>
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                        </tr>
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                        <tr>
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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>
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                          <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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                        </tr>
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                  <center><img src="https://static.igem.org/mediawiki/2016/7/73/T--Ionis_Paris--T4_pool_and_.png" alt=""></center>
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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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        </figure>
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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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                                 <h4>IONIS IGEM</h4>
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                                 <h4>iGEM IONIS</h4>
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                                 <p> We're a group of six different schools from the IONIS Education Group. For this competition we wanted to take advantages of the multiple schools and activity field given by the IONIS education group to create a solid project.</p>
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Revision as of 12:45, 18 October 2016

WHAT IS

Quantifly ?

(VOC)

VOLATILE ORGANIC COMPOUNDS

Nowadays, Volatile Organic Compounds (VOCs) are commonly found pollutants that have been proved 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 requires 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.

If you’ve followed along and done the steps above, you’ve created a good starting place. You’re going to need to make sure your pages are optimized properly.

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