Difference between revisions of "Team:HUST-China/Model"

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     <title>Model</title>
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     <title>Interlab</title>
 
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         @media screen and (max-width: 810px) {
 
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             #section1, #section2, #section3, #section4, #section5 {
 
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                    <li><a href="https://2017.igem.org/Team:HUST-China">HOME</a></li>
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                      <li><a href="https://2017.igem.org/Team:HUST-China">HOME</a></li>
 
                     <li class="dropdown">
 
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                             WETLAB <b class="caret"></b>
 
                             WETLAB <b class="caret"></b>
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                         </ul>
 
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                         <a href="https://2017.igem.org/Team:HUST-China/Model" >
 
                         <a href="https://2017.igem.org/Team:HUST-China/Model" >
 
                             MODEL
 
                             MODEL
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     <div class="tab1" id="section0" style="width:100%;margin-top: 65px;">
 
     <div class="tab1" id="section0" style="width:100%;margin-top: 65px;">
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             <h1 class="tab-h1" ><strong>「Model」</strong></h1>
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             <h1 class="tab-h1" ><strong>「Interlab」</strong></h1>
 
     </div>
 
     </div>
  
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                 <div class="collapse navbar-collapse" id="navbar-collapse">
 
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                     <li class="active"><a href="#section1">Overview</a></li>
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                     <li class="active"><a href="#section1">Introduction</a></li>
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                     <li><a href="#section2">Assumptions</a></li>
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                     <li><a href="#section2">Provenance and Release</a></li>
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                     <li><a href="#section3">Constant & Parameter</a></li>
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                     <li><a href="#section3">Chassis and Safety </a></li>
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                     <li><a href="#section4">Basical Diffusion</a></li>
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                     <li><a href="#section4">Instrument</a></li>
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                     <li><a href="#section5">Recycling</a></li>
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                     <li><a href="#section5">Calibration Protocol</a></li>  
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                     <li><a href="#section6">Sensing and Capture</a></li>
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                     <li><a href="#section6">Cell Culture</a></li>  
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                    <li><a href="#section7">Interlab Result</a></li>
 
                 </ul>
 
                 </ul>
 
                 </div>   
 
                 </div>   
 
                 </div>       
 
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                 <div id="section1" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">     
 
                 <div id="section1" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">     
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                     <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px;margin-top:0;">Overview </h3>
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                     <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px;margin-top:0;">Introduction</h3>
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                     <p>We build an ecosystem level model, in order to see how the Reebot diffuse, sense and capture the lanthanide ion, and be recycled on Si-board. Then we can determine how should we release Reebot, how to recycle the Reebot and how much Reebot do we need to produce the best result. The ecosystem could derive the best strategy to make full use of Reebot.
+
                     <p style="padding:10px">It is significant for synthetic biology to develop a reliable and repeatable measurement, the same to all the other engineering disciplines. We HUST-China have volunteered to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable by measure the expression level of GFP, in order to help the iGEM community collect data about how reliable will these devices turn out to be in labs around the world.
 
                     </p>
 
                     </p>
 
                 </div>
 
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                 <div id="section2" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">  
 
                 <div id="section2" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">  
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                     <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Assumptions</h3>
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                     <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Provenance and Release</h3>
−
                    <p>The variability of temperature has no effect on our Reebot or it could be ignored.</p>
+
                    <h4><strong>①Individuals responsible for conducting InterLab study</strong> </h4>
−
                    <p>Liquid in the container is stationary.</p>
+
                     <div class="table-responsive" style="padding: 10px 100px; text-align: center;">
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                    <p>There is nothing else in the container which can influence Reebot.</p>  
+
−
                </div>
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+
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                <div id="section3" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">       
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                    <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Constant & Parameter</h3>
+
−
                    <p>The description of constant, their values and the references involved in this model are listed in this table.</p>
+
−
 
+
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                         <table class="table table-hover">
 
                           <thead>
 
                           <thead>
 
                               <tr>
 
                               <tr>
−
                                 <th style="text-align: center;">Constant </th>
+
                                 <th style="text-align: center;">Individuals</th>
−
                                 <th style="text-align: center;">Description</th>
+
                                 <th style="text-align: center;">Interlab Part</th>
 
                               </tr>
 
                               </tr>
 
                           </thead>
 
                           </thead>
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                           <tbody>
 
                           <tbody>
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                                  <tr>
+
                              <tr>
−
                                    <td>f<sub>1</sub></td>
+
                                <td>Kangyuan Yu, Haibo Huang, Ziyang Xiao, Shaofeng Liao</td>
−
                                    <td>concentration of Initially Bacteria</td>
+
                                <td>created the devices</td>
−
                                  </tr>
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                              </tr>
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                                  <tr>
+
                              <tr>
−
                                    <td>f<sub>2</sub></td>
+
                                <td>Efan Wang, Long Cheng, HuiPing Shi </td>
−
                                    <td>concentration of Lanthanide ion</td>
+
                                <td>conducted the measurements</td>
−
                                  </tr>
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                              </tr>
−
                                  <tr>
+
                              <tr>
−
                                    <td>f<sub>3</sub></td>
+
                                <td>Efan Wang</td>
−
                                    <td>concentration of Sensed Bacteria</td>
+
                                <td>processed the data</td>
−
                                  </tr>
+
                              </tr>
−
                                  <tr>
+
−
                                    <td>f<sub>4</sub></td>
+
−
                                    <td>concentration of Captured Bacteria</td>
+
−
                                  </tr>
+
−
                                  <tr>
+
−
                                    <td>f<sub>5</sub></td>
+
−
                                    <td>concentration of recycled sensed bacteria </td>
+
−
                                  </tr>
+
−
                                  <tr>
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−
                                    <td>f<sub>6</sub></td>
+
−
                                    <td>concentration of recycled captured bacteria </td>
+
−
                                  </tr>
+
 
                           </tbody>
 
                           </tbody>
 
                         </table>
 
                         </table>
 
                     </div>
 
                     </div>
−
                     <p>The description of parameter, their values and the references involved in this model are listed in the second table.</p>
+
                     <h4><strong> ②Corresponding email</strong> </h4>
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                    <div class="table-responsive " style="padding: 10px 100px; text-align: center; font-size:14px;">
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                               <tr>
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                                 <th style="text-align: center;">Parameter</th>
+
                                 <th style="text-align: center;">Individuals </th>
−
                                 <th style="text-align: center;">Description</th>
+
                                 <th style="text-align: center;">Emails</th>
 
                               </tr>
 
                               </tr>
 
                           </thead>
 
                           </thead>
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                           <tbody>
 
                           <tbody>
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                              <tr>
−
                                    <td>dif</td>
+
                                <td>Efan Wang</td>
−
                                    <td>the diffuse ability of bacteria</td>
+
                                <td>erfan@hust.edu.cn</td>
−
                                  </tr>
+
                              </tr>
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                                  <tr>
+
                              <tr>
−
                                    <td>difl</td>
+
                                <td>Kangyuan Yu</td>
−
                                    <td>the diffuse ability of lanthanide ion</td>
+
                                <td>985930862@qq.com</td>
−
                                  </tr>
+
                              </tr>
−
                                  <tr>
+
                              <tr>
−
                                    <td>deadp</td>
+
                                <td>Haibo Huang </td>
−
                                    <td>dead/birth per minute of bacteria</td>
+
                                <td>u201512127@hust.edu.cn</td>
−
                                  </tr>
+
                              </tr>
−
                                  <tr>
+
                              <tr>
−
                                    <td>vlcx</td>
+
                                <td>Ziyang Xiao</td>
−
                                    <td>velocity of movement on x label</td>
+
                                <td>372657289@qq.com</td>
−
                                  </tr>
+
                              </tr>
−
 
+
                              <tr>
−
                                  <tr>
+
                                <td>Shaofeng Liao</td>
−
                                    <td> vlcy </td>
+
                                <td> 15827233830@qq.com</td>
−
                                    <td>velocity of movement on z label</td>
+
                              </tr>
−
                                  </tr>
+
                              <tr>
−
                                  <tr>
+
                                <td>HuiPing Shi</td>
−
                                    <td>vlcz </td>
+
                                <td>172295915@qq.com</td>
−
                                    <td>concentration of recycled captured bacteria </td>
+
                              </tr>
−
                                  </tr>
+
                              <tr>
−
 
+
                                <td>Long Cheng</td>
−
                                  <tr>
+
                                <td>u201512127@hust.edu.cn</td>
−
                                    <td>krec </td>
+
                              </tr>
−
                                    <td> speed of bacteria recycled on Si-board </td>
+
−
                                  </tr>
+
−
                                  <tr>
+
−
                                    <td>kabcs</td>
+
−
                                    <td>speed of bacteria recycled on Si-board</td>
+
−
                                  </tr>
+
−
 
+
−
                                  <tr>
+
−
                                    <td> rmax </td>
+
−
                                    <td> the amount of bacteria recyclyed on each area of Si-board </td>
+
−
                                  </tr>
+
−
                                  <tr>
+
−
                                    <td>csen</td>
+
−
                                    <td>the lowest concentration lanthanide ion that can be sensed</td>
+
−
                                  </tr>
+
−
 
+
−
                                  <tr>
+
−
                                    <td>ksen </td>
+
−
                                    <td>speed of bacteria sensing the lanthanide ion  </td>
+
−
                                  </tr>
+
−
                                  <tr>
+
−
                                    <td>kcap </td>
+
−
                                    <td> speed of sensed bacteria capturing the lanthanide ion  </td>
+
−
                                  </tr>
+
−
 
+
−
                                  <tr>
+
−
                                    <td>ccap </td>
+
−
                                    <td>the lowest concentration lanthanide ion that can be captured </td>
+
−
                                  </tr>
+
−
                                  <tr>
+
−
                                    <td>LBlever</td>
+
−
                                    <td> The amount of lanthanide ion captured by one bacteria</td>
+
−
                                  </tr>
+
 
                           </tbody>
 
                           </tbody>
 
                         </table>
 
                         </table>
 
                     </div>
 
                     </div>
 +
                </div>       
 +
                <div id="section3" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">       
 +
                    <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Chassis and Safety</h3>
 +
                    <h4><strong> What chassis did you use?</strong> </h4>
 +
                    <p>Escherichia coli DH5alpha</p>
 +
                    <h4><strong> What Biosafety Level is your chassis? </strong> </h4>
 +
                    <p>BSL1</p>
 +
                    <h4><strong>What PPE did you utilize during your experiments? </strong></h4>
 +
                    <p>Tianming gloves</p>
 +
                    <p>Songxinjiujiu labcoats</p>
 
                 </div>
 
                 </div>
 +
                <div id="section4" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">       
 +
                    <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Instrument</h3>
 +
                    <h4><strong> What instrument did you use during your measurements? </strong> </h4>
 +
                    <p>plate reader</p>
 +
                    <h4><strong> Please provide the brand and model of your instrument.</strong> </h4>
 +
                    <p>Flexstation 3</p>
 +
                </div>     
 +
                <div id="section5" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">       
 +
                    <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Calibration Protocol</h3>
  
  
−
                <div id="section4" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">        
+
                    <h4><strong>  A1. Protocol for Optical Density (OD600) Standard Measurement</strong> </h4>
−
                     <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Basical Diffusion</h3>   
+
                     <h5><strong>Did you use pathlength correction during measurement? </strong> </h5>
−
                     <p>Rome was not built in a day. At the beginning, we built a basical diffusion model, just setting the diffusion function in our model.</p>
+
                    <p>Yes</p>
−
                     <p>The function is:</p>
+
                    <h5><strong>Number of flashes per well  </strong> </h5>
−
                     <img class="col-xs-3 col-xs-offset-4" src="https://static.igem.org/mediawiki/2017/5/58/2017_HUST_China_model_equation1.png" />
+
                    <p>6</p>
 +
                    <h5><strong>Orbital averaging (mm)   </strong> </h5>
 +
                     <p>600</p>
 +
                    <h5><strong> What temperature setting did you use during the measurement? </strong> </h5>
 +
                     <p>22℃</p>
 +
                     <h5><strong>What type of 96-well plate did you use? </strong> </h5>
 +
                    <p>Black plate (preferred)</p>
 +
                    <h5><strong> Did your plate have flat-bottomed or round-bottomed wells? </strong> </h5>
 +
                    <p>Flat</p>
  
−
                    <p class="col-sm-12">We release some Reebot to the top of our hypothetical container as initial value of f<sub>1</sub>, and use ‘ode’ tool in MATLAB to run this function. Here is the result:</p>
 
  
−
                     <section class="col-xs-6 col-xs-offset-1">
+
                     <h4><strong> A2. Measurement Steps</strong></h4>
−
                        <figure>
+
                                 <li class="dropdown" style="list-style-type:none;">
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                            <video controls="controls" max-width="600" height="480" poster="https://static.igem.org/mediawiki/2017/d/db/2017_HUST_China_vedio_poster.png" preload="metadata" aria-describedby="full-descript">
+
                                    <button href="#" class="dropdown-toggle" data-toggle="dropdown">
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                                 <source type="video/webm" src="https://static.igem.org/mediawiki/2017/f/fb/2017_HUST_China_model_vedio1.1.webm" />
+
                                        More details <b class="caret"></b>
−
                                <track src="subs/TOS-arabic.srt" kind="subtitles" srclang="ar" label="Arabic" />
+
                                    </button>
−
                                <track src="subs/TOS-japanese.srt" kind="subtitles" srclang="jp" label="Japanese" />
+
                                    <ul class="dropdown-menu container" style="padding: 10px;background:#ccc;">
−
                                <track src="subs/TOS-english.srt" kind="subtitles" srclang="en" label="English" />
+
                                        <li>① Prepare your 96 well plate</li>
−
                                <track src="subs/TOS-turkish.srt" kind="subtitles" srclang="tr" label="Turkish" />
+
                                        <li>②Add 100 μl LUDOX 100 % into wells A1, B1, C1, D1</li>
−
                                <track src="subs/TOS-ukrainian.srt" kind="subtitles" srclang="uk" label="Ukrainian" />
+
                                        <li>③Add 100 μl of dH2O into A2, B2, C2, D2</li>
−
                            </video>
+
                                        <li>④Measure absorbance 600 nm of all samples in all standard measurement modes in instrument</li>
−
                        </figure>
+
                                        <li>⑤Import data into "Abs600" blue cells in provided Excel calibration sheet</li>
−
                    </section>
+
                                    </ul>
 +
                                </li>
  
−
                </div>    
+
                   
−
                <div id="section5" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">   
+
                    <h4><strong>  B1. Protocol for FIuorescein Fluoresence standard curve</strong> </h4>
−
                     <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Recycling</h3>
+
                     <h5><strong>Did you use pathlength correction during measurement? </strong> </h5>
−
                     <p>Then we let our model become more complex and more powerful. We decided using Si-board to recycle the Reebot, since Reebot have Sitag, a protein tag that can combine with Si, once it was sensed. We insert recycling function in our model.</p>
+
                     <p>Yes</p>
−
                     <p>Firstly, we should change the diffusion function:</p>
+
                    <h5><strong>Number of flashes per well  </strong> </h5>
−
                    <img class="col-xs-4 col-xs-offset-4" src="https://static.igem.org/mediawiki/2017/b/b2/2017_HUST_China_model_equation2.png" />
+
                     <p>6</p>
−
                     <p class="col-xs-12" style="text-align: center;">(In the end of all function, the ∂f/∂t will be delta.)</p>
+
                    <h5><strong>What gain setting did you use?  </strong> </h5>
−
                     <p class="col-xs-12">And we set the area of Si-board, if it is in Si-board area:</p>
+
                     <p>Automatic</p>
−
                    <img class="col-sm-5 col-sm-offset-4" src="https://static.igem.org/mediawiki/2017/5/5e/2017_HUST_China_model_equation7.png" />
+
                     <h5><strong> If you used a filter, what light wavelengths did it pass?  </strong> </h5>
−
                     <p class="col-sm-12">Bacteria that combined with Si-board maybe abscess. So taking the abscission into consideration, we should add this function:</p>
+
                     <p>530nm</p>
−
                     <img class="col-xs-4 col-xs-offset-4" src="https://static.igem.org/mediawiki/2017/1/14/2017_HUST_China_model_equation3.png" />
+
                     <h5><strong>Emission wavelength </strong> </h5>
−
                     <p class="col-xs-12">Finally:</p>  
+
                     <p>530nm</p>
−
                     <img class="col-xs-3 col-xs-offset-4" src="https://static.igem.org/mediawiki/2017/9/92/2017_HUST_China_model_equation4.png" />
+
                     <h5><strong> Excitation wavelength </strong> </h5>
−
                     <p class="col-sm-12">Set a 5×5 Si-board on the top and then run the model, the result is:</p>
+
                     <p>485nm</p>
−
                     <section class="col-xs-6 col-xs-offset-1">
+
                     <h5><strong> Fluorescence reading  </strong> </h5>
−
                        <figure>
+
                    <p>Bottom optic</p>
−
                            <video controls="controls" width="600" height="480" poster="https://static.igem.org/mediawiki/2017/d/db/2017_HUST_China_vedio_poster.png" preload="metadata" aria-describedby="full-descript">
+
                    <h5><strong>What type of 96-well plate did you use? </strong> </h5>
−
                                <source type="video/webm" src="https://static.igem.org/mediawiki/2017/a/a5/2017_HUST_China_model_vedio2.1.webm" />
+
                    <p>Black plate (preferred)</p>
−
                                <track src="subs/TOS-arabic.srt" kind="subtitles" srclang="ar" label="Arabic" />
+
                    <h5><strong> Did your plate have flat-bottomed or round-bottomed wells? </strong> </h5>
−
                                <track src="subs/TOS-japanese.srt" kind="subtitles" srclang="jp" label="Japanese" />
+
                    <p>Flat</p>
−
                                <track src="subs/TOS-english.srt" kind="subtitles" srclang="en" label="English" />
+
                    <h5><strong> What temperature setting did you use during the measurement?  </strong> </h5>
−
                                <track src="subs/TOS-turkish.srt" kind="subtitles" srclang="tr" label="Turkish" />
+
                     <p>22℃</p>
−
                                <track src="subs/TOS-ukrainian.srt" kind="subtitles" srclang="uk" label="Ukrainian" />
+
−
                            </video>
+
−
                        </figure>
+
−
                     </section>  
+
  
−
                </div>
+
                    <h4><strong> B2. Measurement Steps</strong></h4>
−
                <div id="section6" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">  
+
                     <h5><strong> Part 1: Prepare the Fluorescein stock solution </strong> </h5>
−
                    <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Sensing and Capture</h3>
+
                            <li class="dropdown" style="list-style-type:none;">
−
                     <p>This is the biggest part of our model. We should take lanthanide ion, sensing and capture into consideration. So, our data doesn’t just contain concentration of bacteria anymore. It now contains four: concentration of initially bacteria (haven’t sensed the lanthanide ion) as f<sub>1</sub>, concentration of lanthanide ion as f<sub>2</sub>, concentration of sensed bacteria（sensed the lanthanide ion but haven’t captured）as f<sub>3</sub> and concentration of captured bacteria (have captured enough lanthanide ion) as f<sub>4</sub>.</p>
+
                                <button href="#" class="dropdown-toggle" data-toggle="dropdown">
−
                    <img class="col-xs-4 col-xs-offset-4" src="https://static.igem.org/mediawiki/2017/5/5b/2017_HUST_China_model_equation5.png" />
+
                                    More details <b class="caret"></b>
−
                    <p class="col-xs-12">And the sensed bacteria will capture the lanthanide ion around it, to simplify the function, we just consider it at two status: no captured and full captured. The function is:</p>
+
                                </button>
−
                    <img class="col-xs-4 col-xs-offset-4" src="https://static.igem.org/mediawiki/2017/a/a0/2017_HUST_China_model_equation6.png" />
+
                                <ul class="dropdown-menu" style="padding: 10px; background:#ccc;">
−
                    <p class="col-xs-12">We should also modify the recycling function. It will only use to sensed bacteria and captured bacteria. </p>
+
                                    <li>①  Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.</li>
−
                    <p class="col-xs-12">Finally, the model is over. Running the model, the result is:</p>
+
                                    <li>②  Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1ml of 1xPBS</li>
−
                    <section class="col-xs-6 col-xs-offset-1">
+
                                    <li>③  Dilute the 2x fluorescein stock solution using 1xPBS to make a 1x fluorescein solution (final concentration is 50 μM).</li>
−
                        <figure>
+
                                    <li>④  *Illustration of serial dilution samples in 96 well plate or cuvettes: value decreases by 2-fold with each column (50% in column 2, 25% in column 3, 12.5% in column 4, etc.)</li>
−
                            <video controls="controls" width="600" height="480" poster="https://static.igem.org/mediawiki/2017/d/db/2017_HUST_China_vedio_poster.png" preload="metadata" aria-describedby="full-descript">
+
                                    <li>⑤Import data into "Abs600" blue cells in provided Excel calibration sheet</li>
−
                                <source type="video/webm" src="https://static.igem.org/mediawiki/2017/6/60/2017_HUST_China_model_vedio3.2.webm" />
+
                                </ul>
−
                                <track src="subs/TOS-arabic.srt" kind="subtitles" srclang="ar" label="Arabic" />
+
                            </li>
−
                                <track src="subs/TOS-japanese.srt" kind="subtitles" srclang="jp" label="Japanese" />
+
                     <h5>Measurement work flow：</h5>
−
                                <track src="subs/TOS-english.srt" kind="subtitles" srclang="en" label="English" />
+
−
                                <track src="subs/TOS-turkish.srt" kind="subtitles" srclang="tr" label="Turkish" />
+
−
                                <track src="subs/TOS-ukrainian.srt" kind="subtitles" srclang="uk" label="Ukrainian" />
+
−
                            </video>
+
−
                        </figure>
+
−
                    </section>
+
−
                     <p class="col-xs-12">For more intuitive, we draw a figure :</p>
+
 
                     <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/>
 
                     <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/>
−
                     <a href="https://static.igem.org/mediawiki/2017/3/39/2017_HUST_China_model_image_part.png" rel="lightbox-demo" title="my caption"><br />
+
                     <a href="https://static.igem.org/mediawiki/2017/f/f8/2017_HUST_China_interlab_img_001.png" rel="lightbox-demo" title="my caption"><br />
−
                         <img title="demo1" src="https://static.igem.org/mediawiki/2017/3/39/2017_HUST_China_model_image_part.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
+
                         <img title="demo1" src="https://static.igem.org/mediawiki/2017/f/f8/2017_HUST_China_interlab_img_001.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
 
                     </a>
 
                     </a>
−
                     <p class="col-xs-12">Put these curve into one figure:</p>  
+
                     <div class="col-xs-12" >
 +
                        <h5><strong>  Part 2: Prepare the serial dilutions of Fluorescein </strong> </h5>
 +
                        <li class="dropdown" style="list-style-type:none;">
 +
                                <button href="#" class="dropdown-toggle" data-toggle="dropdown">
 +
                                    More details <b class="caret"></b>
 +
                                </button>
 +
                                <ul class="dropdown-menu" style="padding: 10px; background:#ccc;">
 +
 
 +
                                        <li>①Add 100 μl of 1xPBS into wells A2, B2, C2, D2....A12, B12, C12, D12.</li>
 +
                                        <li>②Add 200 μl of 1x Fluorescein stock solution into A1, B1, C1, D1.</li>
 +
                                        <li>③  Transfer 100 μl of Fluorescein stock solution from A1 into A2.</li>
 +
                                        <li>④  Mix A2 by pipetting up and down 3x and transfer 100 μl into A3.</li>
 +
                                        <li>⑤  Mix A3 by pipetting up and down 3x and transfer 100 μl into A4.</li>
 +
                                        <li>⑥  Mix A4 by pipetting up and down 3x and transfer 100 μl into A5.</li>
 +
                                        <li>⑦  Mix A5 by pipetting up and down 3x and transfer 100 μl into A6.</li>
 +
                                        <li>⑧  Mix A6 by pipetting up and down 3x and transfer 100 μl into A7.</li>
 +
                                        <li>⑨  Mix A7 by pipetting up and down 3x and transfer 100 μl into A8.</li>
 +
                                        <li>⑩  Mix A8 by pipetting up and down 3x and transfer 100 μl into A9.</li>
 +
                                        <li>⑪  Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.</li>
 +
                                        <li>⑫  Mix A10 by pipetting up and down 3x and transfer 100 μl into A11.</li>
 +
                                        <li>⑬  Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.</li>
 +
                                        <li>⑭  TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.</li>
 +
                                        <li>⑮  Repeat dilution series for rows B, C, D.
 +
                                        <li>⑯  Measure fluorescence of all samples in all standard measurement modes in instrument.
 +
                                </ul>
 +
                            </li>
 +
 
 +
                    </div>
 +
   
 +
                </div>
 +
                <div id="section6" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">   
 +
                    <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px;margin-top:0;">Cell Culture Setup and Measurement</h3>
 
                     <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/>
 
                     <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/>
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                     <a href="https://static.igem.org/mediawiki/2017/6/66/2017_HUST_Chian_model_image_all.png" rel="lightbox-demo" title="my caption"><br />
+
                     <a href="https://static.igem.org/mediawiki/2017/e/e6/2017_HUST_China_interlab_img_002.png" rel="lightbox-demo" title="my caption"><br />
−
                         <img title="demo1" src="https://static.igem.org/mediawiki/2017/6/66/2017_HUST_Chian_model_image_all.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
+
                         <img title="demo1" src="https://static.igem.org/mediawiki/2017/e/e6/2017_HUST_China_interlab_img_002.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
 
                     </a>
 
                     </a>
−
                    <p class="col-xs-12">It’s clearly that there are three stages during the whole simulation. The first stage is sensing, second stage is capturing and third is recycling.</p>
 
 
                     <div class="col-xs-12">
 
                     <div class="col-xs-12">
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                         <h4 style="color:#97477c"><strong>Result:</strong></h4>
+
                         <h5><strong>Transformation:</strong> </h5>
−
                        <p>Firstly, we run a model which is: there are enough concentration of lanthanide ion in the consider, and we put some Reebot on the top. Here is the video:</p>
+
                            <ul class="yuandian">
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                         <section class="col-xs-6 col-xs-offset-1">
+
                                <li> control</li>
−
                            <figure>
+
                                <li> Negative control</li>
−
                                 <video controls="controls" width="600" height="480" poster="https://static.igem.org/mediawiki/2017/d/db/2017_HUST_China_vedio_poster.png" preload="metadata" aria-describedby="full-descript">
+
                                <li>Test Device 1: J23101+I13504</li>
−
                                     <source type="video/webm" src="https://static.igem.org/mediawiki/2017/6/60/2017_HUST_China_model_vedio3.2.webm" />
+
                                <li>Test Device 2: J23106+I13504</li>
−
                                     <track src="subs/TOS-arabic.srt" kind="subtitles" srclang="ar" label="Arabic" />
+
                                <li>Test Device 3: J23117+I13504</li>
−
                                    <track src="subs/TOS-japanese.srt" kind="subtitles" srclang="jp" label="Japanese" />
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                                <li>Test Device 4: J23101.BCD2.E0040.B0015</li>
−
                                    <track src="subs/TOS-english.srt" kind="subtitles" srclang="en" label="English" />
+
                                <li>Test Device 5: J23106.BCD2.E0040.B0015</li>
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                                     <track src="subs/TOS-turkish.srt" kind="subtitles" srclang="tr" label="Turkish" />
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                                <li>Test Device 6: J23117.BCD2.E0040.B0015</li>
−
                                     <track src="subs/TOS-ukrainian.srt" kind="subtitles" srclang="uk" label="Ukrainian" />
+
                            </ul>
−
                                 </video>
+
                         <h5><strong>Cell growth:</strong> </h5>
−
                             </figure>
+
                        <li class="dropdown" style="list-style-type:none;">
−
                         </section>
+
                                 <button href="#" class="dropdown-toggle" data-toggle="dropdown">
 +
                                    More details <b class="caret"></b>
 +
                                </button>
 +
                                <ul class="dropdown-menu" style="padding: 10px; background:#ccc;">
 +
 
 +
                                     <li> ①    Pick 2 colonies from each of plate and inoculate each in 5-10 mL LB medium + Chloramphenicol (For antibiotic concentrations, please follow these guidelines:<a href="http://parts.igem.org/Help:Protocols/Antibiotic_Stocks">http://parts.igem.org/Help:Protocols/Antibiotic_Stocks).
 +
                                    </a>
 +
                                     </li>
 +
                                    <li>②  Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.</li>
 +
                                    <li>③  With 8 total devices (including controls) and 2 colonies per device, you should have 16 cultures.</li>
 +
 
 +
                                </ul>
 +
                            </li>
 +
                        <h5><strong>Cell growth, sampling, and assay</strong> </h5>
 +
                        <li class="dropdown" style="list-style-type:none;">
 +
                                <button href="#" class="dropdown-toggle" data-toggle="dropdown">
 +
                                    More details <b class="caret"></b>
 +
                                </button>
 +
                                <ul class="dropdown-menu" style="padding: 10px; background:#ccc;">
 +
 
 +
                                    <li>①  Set your instrument to read Abs600 (as OD calibration setting)</li>
 +
                                     <li>②  Measure Abs600 of the overnight cultures</li>
 +
                                    <li>③  Import data into blue cells in Excel Dilultion Calculation sheets provided</li>
 +
                                    <li>④  Dilute the cultures to a target Abs600 of 0.02 (see the volume of preloading culture and media in Excel Dilution Calculation sheets) in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube.</li>
 +
                                     <li>⑤  Remove 500uL of each culture for your zero time point and hold these samples on ice. (You should have 16 sample tubes from the time=0 time point)</li>
 +
                                    <li>⑥  Incubate the cultures at 37°C and 220 rpm.</li>
 +
                                    <li>⑦  Remove 500uL samples of each culture after 2, 4 and 6 hours of growth. Keep samples on ice while completing the additional time points. You should have 16 sample tubes for each time point.</li>
 +
                                    <li>⑧  Set up your four measurement plates: For colony #1 culture, pipet 100 uL samples into wells A, B, C and D of the row for that device. For colony #2, pipet 100uL samples into wells E, F, G, and H of the row for that device.</li>
 +
                                    <li>⑨  Read your plates, taking care to use the exact same settings used for your fluorescein measurement.</li>
 +
 
 +
 
 +
                                 </ul>
 +
                             </li>
 +
                         <h5><strong>The initial OD600 measurement of our overnight cultures.</strong> </h5>
 +
                        <img title="demo1" src="https://static.igem.org/mediawiki/2017/1/1a/2017_HUST_China_interlab_img_table1.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
 
                     </div>
 
                     </div>
 
                     <div class="col-xs-12">
 
                     <div class="col-xs-12">
−
                         <p> From the video, we can see that once we put Reebot into the container, the Reebot quickly diffuse. At the same time, Reebot sense the lanthanide ion around them, and express more LBT (lanthanide binding tag), sitag on their surface. Then, the LBT start to capture the lanthanide ion in the environment. As time goes by, Reebot have captured more and more lanthanide. At the beginning, the concentration of lanthanide ion is 0.02，and now, it is 0.006.</p>
+
                        <h5><strong>What type of media did you use for this step? </strong> </h5>
−
                         <p>When t = 60, we set a 6*8 Si-board on the top. The sensed bacteria and captured bacteria are recycling to the Si-board. You can see it clearly that the edge of Si-board recycled more Reebot. In the end, many bacteria are recycled to the Si-board, but there are still many Reebot in the environment. The reason is concentration gradient caused by Si-board isn’t large enough so that the diffusion isn’t effective. But keeping the Si-board here doesn’t cost much, so we can stand it.</p>
+
                         <p>Luria Bertani </p>
−
                         <p>In order to show you more details, we sum each areas’ concentration and put them into on figure:</p>
+
                        <h5><strong>What type of vessel or container did you use to grow your cells? </strong> </h5>
 +
                         <p>50 ml Falcon tube</p>
 +
                        <h5><strong>What temperature setting did you use during the measurement?</strong> </h5>
 +
                        <p>22℃</p>
 +
                        <h5><strong>What type of 96-well plates did you use?</strong> </h5>
 +
                        <p></p>
 +
                        <h5><strong>Black plates with transparent/clear bottom (preferred)</strong> </h5>
 +
                         <p>Flat</p>
 +
                        <h5><strong>Measurement </strong> </h5>
 +
                        <ul class="yuandian">
 +
                                <li>Measure OD and fluorescence of all samples</li>
 +
                                <li>Import data into blue cells in Excel (cell measurement) sheets provided</li>
 +
                        </ul>
 +
                        <h5><strong>Suggested Plate Layout for 96-well Plate</strong> </h5>
 
                         <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/>
 
                         <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/>
−
                         <a href="https://static.igem.org/mediawiki/2017/6/66/2017_HUST_Chian_model_image_all.png" rel="lightbox-demo" title="my caption"><br />
+
                         <a href="https://static.igem.org/mediawiki/2017/8/87/2017_HUST_China_interlab_img_003.png" rel="lightbox-demo" title="my caption"><br />
−
                             <img title="demo1" src="https://static.igem.org/mediawiki/2017/6/66/2017_HUST_Chian_model_image_all.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
+
                             <img title="demo1" src="https://static.igem.org/mediawiki/2017/8/87/2017_HUST_China_interlab_img_003.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
 
                         </a>
 
                         </a>
−
                    </div>
 
−
                    <div class="col-xs-12">
 
−
                        <p> From the figure, we can see lot of things. In the first 60 units of time, most Reebot quickly captured enough bacteria, the concentration of initial bacteria and sensed bacteria are very low. Then, the Si-board set, Reebot begin to recycle. At first, in the figure, Reebot recycle at about a same speed. Then recycle speed is slower and slower. </p>
 
−
                        <p>This model simulate the ordinary use of Reebot. the result can show us the detail of process, we can determine how much Reebot to release and recycle for how long upon that.</p>
 
−
                        <p>Then, we run an other simulation to show the intelligence of Reebot. In this simulation, there was no lanthanide ion at the beginning, we put lanthanide ion at some certain time.</p>
 
−
                        <p>The video and figure is here.</p>
 
−
                        <section class="col-xs-6 col-xs-offset-1">
 
−
                            <figure>
 
−
                                <video controls="controls" width="600" height="480" poster="https://static.igem.org/mediawiki/2017/d/db/2017_HUST_China_vedio_poster.png" preload="metadata" aria-describedby="full-descript">
 
−
                                    <source type="video/webm" src="https://static.igem.org/mediawiki/2017/a/ab/2017_HUST_China_model_vedio4.2.webm" />
 
−
                                    <track src="subs/TOS-arabic.srt" kind="subtitles" srclang="ar" label="Arabic" />
 
−
                                    <track src="subs/TOS-japanese.srt" kind="subtitles" srclang="jp" label="Japanese" />
 
−
                                    <track src="subs/TOS-english.srt" kind="subtitles" srclang="en" label="English" />
 
−
                                    <track src="subs/TOS-turkish.srt" kind="subtitles" srclang="tr" label="Turkish" />
 
−
                                    <track src="subs/TOS-ukrainian.srt" kind="subtitles" srclang="uk" label="Ukrainian" />
 
−
                                </video>
 
−
                            </figure>
 
−
                        </section>
 
−
                    </div>
 
−
                    <div class="col-xs-12">
 
−
                        <p>At the beginning, we release Reebot on the top. While there is no lanthanide ion in environment, so Reebot keep the initial status, and diffuse in the container. When passing through 40 units of time, there are some lanthanide ion release at the bottom of container. From the video, Reebot take a quick reaction, capturing the lanthanide ion and recycling. When it passes 180 units of time, other lanthanide ion release at another position in bottom. Reebot successfully capture them too.</p>
 
−
                        <p>It’s also our project’s future plan. Once we achieve it, we can release Reebot in factory’s pool. If there is no lanthanide ion, Reebot just live as a normal escherichia coli. And once lanthanide ion emerge, Reebot can sense and capture it, then Reebot combine with Si-board. </p>
 
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
 +
                <div id="section7" style="border: solid 1px #666; margin:5px; padding:5px;border-radius:10px;overflow: hidden;">   
 +
                    <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px;margin-top:0;">Interlab Resultn</h3>
 +
                    <h5 class="col-xs-12"><strong>OD600 Reference Point</strong> </h5>
 +
                    <img title="demo1" src="https://static.igem.org/mediawiki/2017/5/52/2017_HUST_China_interlab_img_table2.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
 +
                    <h5 class="col-xs-12"><strong>Fluorescence standard curve</strong> </h5>
 +
                    <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/>
 +
                        <a href="https://static.igem.org/mediawiki/2017/a/a1/2017_HUST_China_interlab_img_table3.png" rel="lightbox-demo" title="my caption"><br />
 +
                            <img title="demo1" src="https://static.igem.org/mediawiki/2017/a/a1/2017_HUST_China_interlab_img_table3.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
 +
                        </a>
 +
                    <p class="col-xs-12"><strong>NOTE: 50uM Sample exceeds the range of measurements</strong> </p>
 +
                    <h5 class="col-xs-12"><strong>OD600 Reference Point</strong> </h5>
 +
                    <img title="demo1" src="https://static.igem.org/mediawiki/2017/d/d1/2017_HUST_China_interlab_img_table4.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
 +
                    <img title="demo1" src="https://static.igem.org/mediawiki/2017/d/df/2017_HUST_China_interlab_img_table5.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
 +
                    <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/>
 +
                        <a href="https://static.igem.org/mediawiki/2017/b/b3/2017_HUST_China_interlab_img_table6.png" rel="lightbox-demo" title="my caption"><br />
 +
                            <img title="demo1" src="https://static.igem.org/mediawiki/2017/b/b3/2017_HUST_China_interlab_img_table6.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
 +
                        </a>
 +
                    <p class="col-xs-12"><strong>Final scaling level determined from medium-high points likely to be less impacted by saturation or pipetting error.</strong> </p>
 +
                      <p class="col-xs-12"><strong>If needed, you can shift which points are used, but it is likely better to correct instrument settings and protocol.</strong> </p>
 +
                    <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/>
 +
                        <a href="https://static.igem.org/mediawiki/2017/b/b8/2017_HUST_China_interlab_img_table7.png" rel="lightbox-demo" title="my caption"><br />
 +
                            <img title="demo1" src="https://static.igem.org/mediawiki/2017/b/b8/2017_HUST_China_interlab_img_table7.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
 +
                        </a>
 +
                    <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/>
 +
                        <a href="https://static.igem.org/mediawiki/2017/e/ea/2017_HUST_China_interlab_img_table8.png" title="my caption"><br />
 +
                            <img title="demo1" src="https://static.igem.org/mediawiki/2017/e/ea/2017_HUST_China_interlab_img_table8.png" alt="demo1" class="col-xs-8 col-xs-offset-2">
 +
                        </a>
 +
                    <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/>
 +
                        <a href="https://static.igem.org/mediawiki/2017/d/d2/2017_HUST_China_interlab_img_table9.png" rel="lightbox-demo" title="my caption"><br />
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Revision as of 16:10, 28 October 2017

Interlab

「Interlab」

Introduction

It is significant for synthetic biology to develop a reliable and repeatable measurement, the same to all the other engineering disciplines. We HUST-China have volunteered to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable by measure the expression level of GFP, in order to help the iGEM community collect data about how reliable will these devices turn out to be in labs around the world.

Provenance and Release

①Individuals responsible for conducting InterLab study

Individuals Interlab Part
Kangyuan Yu, Haibo Huang, Ziyang Xiao, Shaofeng Liao created the devices
Efan Wang, Long Cheng, HuiPing Shi conducted the measurements
Efan Wang processed the data

②Corresponding email

Individuals Emails
Efan Wang erfan@hust.edu.cn
Kangyuan Yu 985930862@qq.com
Haibo Huang u201512127@hust.edu.cn
Ziyang Xiao 372657289@qq.com
Shaofeng Liao 15827233830@qq.com
HuiPing Shi 172295915@qq.com
Long Cheng u201512127@hust.edu.cn

Chassis and Safety

What chassis did you use?

Escherichia coli DH5alpha

What Biosafety Level is your chassis?

BSL1

What PPE did you utilize during your experiments?

Tianming gloves

Songxinjiujiu labcoats

Instrument

What instrument did you use during your measurements?

plate reader

Please provide the brand and model of your instrument.

Flexstation 3

Calibration Protocol

A1. Protocol for Optical Density (OD600) Standard Measurement

Did you use pathlength correction during measurement?

Yes

Number of flashes per well

6

Orbital averaging (mm)

600

What temperature setting did you use during the measurement?

22℃

What type of 96-well plate did you use?

Black plate (preferred)

Did your plate have flat-bottomed or round-bottomed wells?

Flat

A2. Measurement Steps

    • ① Prepare your 96 well plate
    • ②Add 100 μl LUDOX 100 % into wells A1, B1, C1, D1
    • ③Add 100 μl of dH2O into A2, B2, C2, D2
    • ④Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
    • ⑤Import data into "Abs600" blue cells in provided Excel calibration sheet

    • B1. Protocol for FIuorescein Fluoresence standard curve

    Did you use pathlength correction during measurement?

    Yes

    Number of flashes per well

    6

    What gain setting did you use?

    Automatic

    If you used a filter, what light wavelengths did it pass?

    530nm

    Emission wavelength

    530nm

    Excitation wavelength

    485nm

    Fluorescence reading

    Bottom optic

    What type of 96-well plate did you use?

    Black plate (preferred)

    Did your plate have flat-bottomed or round-bottomed wells?

    Flat

    What temperature setting did you use during the measurement?

    22℃

    B2. Measurement Steps

    Part 1: Prepare the Fluorescein stock solution
    • ① Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.
    • ② Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1ml of 1xPBS
    • ③ Dilute the 2x fluorescein stock solution using 1xPBS to make a 1x fluorescein solution (final concentration is 50 μM).
    • ④ *Illustration of serial dilution samples in 96 well plate or cuvettes: value decreases by 2-fold with each column (50% in column 2, 25% in column 3, 12.5% in column 4, etc.)
    • ⑤Import data into "Abs600" blue cells in provided Excel calibration sheet
    • Measurement work flow：

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    demo1
    Part 2: Prepare the serial dilutions of Fluorescein
    • ①Add 100 μl of 1xPBS into wells A2, B2, C2, D2....A12, B12, C12, D12.
    • ②Add 200 μl of 1x Fluorescein stock solution into A1, B1, C1, D1.
    • ③ Transfer 100 μl of Fluorescein stock solution from A1 into A2.
    • ④ Mix A2 by pipetting up and down 3x and transfer 100 μl into A3.
    • ⑤ Mix A3 by pipetting up and down 3x and transfer 100 μl into A4.
    • ⑥ Mix A4 by pipetting up and down 3x and transfer 100 μl into A5.
    • ⑦ Mix A5 by pipetting up and down 3x and transfer 100 μl into A6.
    • ⑧ Mix A6 by pipetting up and down 3x and transfer 100 μl into A7.
    • ⑨ Mix A7 by pipetting up and down 3x and transfer 100 μl into A8.
    • ⑩ Mix A8 by pipetting up and down 3x and transfer 100 μl into A9.
    • ⑪ Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.
    • ⑫ Mix A10 by pipetting up and down 3x and transfer 100 μl into A11.
    • ⑬ Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.
    • ⑭ TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.
    • ⑮ Repeat dilution series for rows B, C, D.
    • ⑯ Measure fluorescence of all samples in all standard measurement modes in instrument.

    • Cell Culture Setup and Measurement

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    demo1
    Transformation:
    • control
    • Negative control
    • Test Device 1: J23101+I13504
    • Test Device 2: J23106+I13504
    • Test Device 3: J23117+I13504
    • Test Device 4: J23101.BCD2.E0040.B0015
    • Test Device 5: J23106.BCD2.E0040.B0015
    • Test Device 6: J23117.BCD2.E0040.B0015
    Cell growth:
    • ① Pick 2 colonies from each of plate and inoculate each in 5-10 mL LB medium + Chloramphenicol (For antibiotic concentrations, please follow these guidelines:http://parts.igem.org/Help:Protocols/Antibiotic_Stocks).
    • ② Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.
    • ③ With 8 total devices (including controls) and 2 colonies per device, you should have 16 cultures.
    • Cell growth, sampling, and assay
    • ① Set your instrument to read Abs600 (as OD calibration setting)
    • ② Measure Abs600 of the overnight cultures
    • ③ Import data into blue cells in Excel Dilultion Calculation sheets provided
    • ④ Dilute the cultures to a target Abs600 of 0.02 (see the volume of preloading culture and media in Excel Dilution Calculation sheets) in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube.
    • ⑤ Remove 500uL of each culture for your zero time point and hold these samples on ice. (You should have 16 sample tubes from the time=0 time point)
    • ⑥ Incubate the cultures at 37°C and 220 rpm.
    • ⑦ Remove 500uL samples of each culture after 2, 4 and 6 hours of growth. Keep samples on ice while completing the additional time points. You should have 16 sample tubes for each time point.
    • ⑧ Set up your four measurement plates: For colony #1 culture, pipet 100 uL samples into wells A, B, C and D of the row for that device. For colony #2, pipet 100uL samples into wells E, F, G, and H of the row for that device.
    • ⑨ Read your plates, taking care to use the exact same settings used for your fluorescein measurement.
    • The initial OD600 measurement of our overnight cultures.
    demo1
    What type of media did you use for this step?

    Luria Bertani

    What type of vessel or container did you use to grow your cells?

    50 ml Falcon tube

    What temperature setting did you use during the measurement?

    22℃

    What type of 96-well plates did you use?

    Black plates with transparent/clear bottom (preferred)

    Flat

    Measurement
    • Measure OD and fluorescence of all samples
    • Import data into blue cells in Excel (cell measurement) sheets provided
    Suggested Plate Layout for 96-well Plate

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    demo1

    Interlab Resultn

    OD600 Reference Point
    demo1
    Fluorescence standard curve

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    demo1

    NOTE: 50uM Sample exceeds the range of measurements

    OD600 Reference Point
    demo1 demo1

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    demo1

    Final scaling level determined from medium-high points likely to be less impacted by saturation or pipetting error.

    If needed, you can shift which points are used, but it is likely better to correct instrument settings and protocol.

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    demo1

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    demo1

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    demo1

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    demo1

    Acknowledgments: