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

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     <title>Interlab</title>
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     <title>Model</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>
 
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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>「Interlab」</strong></h1>
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             <h1 class="tab-h1" ><strong>「Model」</strong></h1>
 
     </div>
 
     </div>
  
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                     <li class="active"><a href="#section1">Introduction</a></li>
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                     <li class="active"><a href="#section1">Overview</a></li>
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                     <li><a href="#section2">Provenance and Release</a></li>
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                     <li><a href="#section2">Assumptions</a></li>
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                     <li><a href="#section3">Chassis and Safety </a></li>
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                     <li><a href="#section3">Constant & Parameter</a></li>
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                     <li><a href="#section4">Instrument</a></li>
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                     <li><a href="#section4">Basical Diffusion</a></li>
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                     <li><a href="#section5">Calibration Protocol</a></li>  
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                     <li><a href="#section5">Recycling</a></li>
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                     <li><a href="#section6">Cell Culture</a></li>  
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                     <li><a href="#section6">Sensing and Capture</a></li>
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                    <li><a href="#section7">Interlab Result</a></li>
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                 </ul>
 
                 </ul>
 
                 </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;">Introduction</h3>
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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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                     <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>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>
 
                     </p>
 
                 </div>
 
                 </div>
 +
 
                 <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;">  
−
                     <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Provenance and Release</h3>
+
                     <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Assumptions</h3>
−
                    <h4><strong>①Individuals responsible for conducting InterLab study</strong> </h4>
+
                    <p>The variability of temperature has no effect on our Reebot or it could be ignored.</p>
−
                     <div class="table-responsive" style="padding: 10px 100px; text-align: center;">
+
                    <p>Liquid in the container is stationary.</p>
 +
                    <p>There is nothing else in the container which can influence Reebot.</p>  
 +
                </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;">Constant & Parameter</h3>
 +
                    <p>The description of constant, their values and the references involved in this model are listed in this table.</p>
 +
 
 +
                     <div class="table-responsive " style="padding: 10px 100px; text-align: center; font-size:14px;">
 
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                           <thead>
 
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                               <tr>
 
                               <tr>
−
                                 <th style="text-align: center;">Individuals</th>
+
                                 <th style="text-align: center;">Constant </th>
−
                                 <th style="text-align: center;">Interlab Part</th>
+
                                 <th style="text-align: center;">Description</th>
 
                               </tr>
 
                               </tr>
 
                           </thead>
 
                           </thead>
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 +
 
                           <tbody>
 
                           <tbody>
−
                              <tr>
+
                                  <tr>
−
                                <td>Kangyuan Yu, Haibo Huang, Ziyang Xiao, Shaofeng Liao</td>
+
                                    <td>f<sub>1</sub></td>
−
                                <td>created the devices</td>
+
                                    <td>concentration of Initially Bacteria</td>
−
                              </tr>
+
                                  </tr>
−
                              <tr>
+
                                  <tr>
−
                                <td>Efan Wang, Long Cheng, HuiPing Shi </td>
+
                                    <td>f<sub>2</sub></td>
−
                                <td>conducted the measurements</td>
+
                                    <td>concentration of Lanthanide ion</td>
−
                              </tr>
+
                                  </tr>
−
                              <tr>
+
                                  <tr>
−
                                <td>Efan Wang</td>
+
                                    <td>f<sub>3</sub></td>
−
                                <td>processed the data</td>
+
                                    <td>concentration of Sensed Bacteria</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>
 +
                                    <td>f<sub>6</sub></td>
 +
                                    <td>concentration of recycled captured bacteria </td>
 +
                                  </tr>
 
                           </tbody>
 
                           </tbody>
 
                         </table>
 
                         </table>
 
                     </div>
 
                     </div>
−
                     <h4><strong> ②Corresponding email</strong> </h4>
+
                     <p>The description of parameter, their values and the references involved in this model are listed in the second table.</p>
−
                    <div class="table-responsive" style="padding: 10px 100px; text-align: center;">
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                               <tr>
 
                               <tr>
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                                 <th style="text-align: center;">Individuals </th>
+
                                 <th style="text-align: center;">Parameter</th>
−
                                 <th style="text-align: center;">Emails</th>
+
                                 <th style="text-align: center;">Description</th>
 
                               </tr>
 
                               </tr>
 
                           </thead>
 
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                           <tbody>
 
                           <tbody>
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                              <tr>
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                                  <tr>
−
                                <td>Efan Wang</td>
+
                                    <td>dif</td>
−
                                <td>erfan@hust.edu.cn</td>
+
                                    <td>the diffuse ability of bacteria</td>
−
                              </tr>
+
                                  </tr>
−
                              <tr>
+
                                  <tr>
−
                                <td>Kangyuan Yu</td>
+
                                    <td>difl</td>
−
                                <td>985930862@qq.com</td>
+
                                    <td>the diffuse ability of lanthanide ion</td>
−
                              </tr>
+
                                  </tr>
−
                              <tr>
+
                                  <tr>
−
                                <td>Haibo Huang </td>
+
                                    <td>deadp</td>
−
                                <td>u201512127@hust.edu.cn</td>
+
                                    <td>dead/birth per minute of bacteria</td>
−
                              </tr>
+
                                  </tr>
−
                              <tr>
+
                                  <tr>
−
                                <td>Ziyang Xiao</td>
+
                                    <td>vlcx</td>
−
                                <td>372657289@qq.com</td>
+
                                    <td>velocity of movement on x label</td>
−
                              </tr>
+
                                  </tr>
−
                              <tr>
+
 
−
                                <td>Shaofeng Liao</td>
+
                                  <tr>
−
                                <td> 15827233830@qq.com</td>
+
                                    <td> vlcy </td>
−
                              </tr>
+
                                    <td>velocity of movement on z label</td>
−
                              <tr>
+
                                  </tr>
−
                                <td>HuiPing Shi</td>
+
                                  <tr>
−
                                <td>172295915@qq.com</td>
+
                                    <td>vlcz </td>
−
                              </tr>
+
                                    <td>concentration of recycled captured bacteria </td>
−
                              <tr>
+
                                  </tr>
−
                                <td>Long Cheng</td>
+
 
−
                                <td>u201512127@hust.edu.cn</td>
+
                                  <tr>
−
                              </tr>
+
                                    <td>krec </td>
 +
                                    <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>
 
  
  
−
                    <h4><strong>  A1. Protocol for Optical Density (OD600) Standard Measurement</strong> </h4>
+
                <div id="section4" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">        
−
                     <h5><strong>Did you use pathlength correction during measurement? </strong> </h5>
+
                     <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Basical Diffusion</h3>   
−
                    <p>Yes</p>
+
                     <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>
−
                    <h5><strong>Number of flashes per well  </strong> </h5>
+
                     <p>The function is:</p>
−
                    <p>6</p>
+
                     <img class="col-xs-3 col-xs-offset-4" src="https://static.igem.org/mediawiki/2017/5/58/2017_HUST_China_model_equation1.png" />
−
                    <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>
  
−
                     <h4><strong> A2. Measurement Steps</strong></h4>
+
                     <section class="col-xs-6 col-xs-offset-1">
−
                                 <li class="dropdown" style="list-style-type:none;">
+
                        <figure>
−
                                    <button href="#" class="dropdown-toggle" data-toggle="dropdown">
+
                            <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">
−
                                        More details <b class="caret"></b>
+
                                 <source type="video/webm" src="https://static.igem.org/mediawiki/2017/f/fb/2017_HUST_China_model_vedio1.1.webm" />
−
                                    </button>
+
                                <track src="subs/TOS-arabic.srt" kind="subtitles" srclang="ar" label="Arabic" />
−
                                    <ul class="dropdown-menu container" style="padding: 10px;background:#ccc;">
+
                                <track src="subs/TOS-japanese.srt" kind="subtitles" srclang="jp" label="Japanese" />
−
                                        <li>① Prepare your 96 well plate</li>
+
                                <track src="subs/TOS-english.srt" kind="subtitles" srclang="en" label="English" />
−
                                        <li>②Add 100 μl LUDOX 100 % into wells A1, B1, C1, D1</li>
+
                                <track src="subs/TOS-turkish.srt" kind="subtitles" srclang="tr" label="Turkish" />
−
                                        <li>③Add 100 μl of dH2O into A2, B2, C2, D2</li>
+
                                <track src="subs/TOS-ukrainian.srt" kind="subtitles" srclang="uk" label="Ukrainian" />
−
                                        <li>④Measure absorbance 600 nm of all samples in all standard measurement modes in instrument</li>
+
                            </video>
−
                                        <li>⑤Import data into "Abs600" blue cells in provided Excel calibration sheet</li>
+
                        </figure>
−
                                    </ul>
+
                    </section>
−
                                </li>
+
  
−
                   
+
                </div>    
−
                    <h4><strong>  B1. Protocol for FIuorescein Fluoresence standard curve</strong> </h4>
+
                <div id="section5" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">   
−
                     <h5><strong>Did you use pathlength correction during measurement? </strong> </h5>
+
                     <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Recycling</h3>
−
                     <p>Yes</p>
+
                     <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>
−
                    <h5><strong>Number of flashes per well  </strong> </h5>
+
                     <p>Firstly, we should change the diffusion function:</p>
−
                     <p>6</p>
+
                    <img class="col-xs-4 col-xs-offset-4" src="https://static.igem.org/mediawiki/2017/b/b2/2017_HUST_China_model_equation2.png" />
−
                    <h5><strong>What gain setting did you use?  </strong> </h5>
+
                     <p class="col-xs-12" style="text-align: center;">(In the end of all function, the ∂f/∂t will be delta.)</p>
−
                     <p>Automatic</p>
+
                     <p class="col-xs-12">And we set the area of Si-board, if it is in Si-board area:</p>
−
                     <h5><strong> If you used a filter, what light wavelengths did it pass?  </strong> </h5>
+
                    <img class="col-sm-5 col-sm-offset-4" src="https://static.igem.org/mediawiki/2017/5/5e/2017_HUST_China_model_equation7.png" />
−
                     <p>530nm</p>
+
                     <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>
−
                     <h5><strong>Emission wavelength </strong> </h5>
+
                     <img class="col-xs-4 col-xs-offset-4" src="https://static.igem.org/mediawiki/2017/1/14/2017_HUST_China_model_equation3.png" />
−
                     <p>530nm</p>
+
                     <p class="col-xs-12">Finally:</p>  
−
                     <h5><strong> Excitation wavelength </strong> </h5>
+
                     <img class="col-xs-3 col-xs-offset-4" src="https://static.igem.org/mediawiki/2017/9/92/2017_HUST_China_model_equation4.png" />
−
                     <p>485nm</p>
+
                     <p class="col-sm-12">Set a 5×5 Si-board on the top and then run the model, the result is:</p>
−
                     <h5><strong> Fluorescence reading  </strong> </h5>
+
                     <section class="col-xs-6 col-xs-offset-1">
−
                    <p>Bottom optic</p>
+
                        <figure>
−
                    <h5><strong>What type of 96-well plate did you use? </strong> </h5>
+
                            <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">
−
                    <p>Black plate (preferred)</p>
+
                                <source type="video/webm" src="https://static.igem.org/mediawiki/2017/a/a5/2017_HUST_China_model_vedio2.1.webm" />
−
                    <h5><strong> Did your plate have flat-bottomed or round-bottomed wells? </strong> </h5>
+
                                <track src="subs/TOS-arabic.srt" kind="subtitles" srclang="ar" label="Arabic" />
−
                    <p>Flat</p>
+
                                <track src="subs/TOS-japanese.srt" kind="subtitles" srclang="jp" label="Japanese" />
−
                    <h5><strong> What temperature setting did you use during the measurement?  </strong> </h5>
+
                                <track src="subs/TOS-english.srt" kind="subtitles" srclang="en" label="English" />
−
                     <p>22℃</p>
+
                                <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>  
  
−
                    <h4><strong> B2. Measurement Steps</strong></h4>
+
                </div>
−
                     <h5><strong> Part 1: Prepare the Fluorescein stock solution </strong> </h5>
+
                <div id="section6" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;">  
−
                            <li class="dropdown" style="list-style-type:none;">
+
                    <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Sensing and Capture</h3>
−
                                <button href="#" class="dropdown-toggle" data-toggle="dropdown">
+
                     <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>
−
                                    More details <b class="caret"></b>
+
                    <img class="col-xs-4 col-xs-offset-4" src="https://static.igem.org/mediawiki/2017/5/5b/2017_HUST_China_model_equation5.png" />
−
                                </button>
+
                    <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>
−
                                <ul class="dropdown-menu" style="padding: 10px; background:#ccc;">
+
                    <img class="col-xs-4 col-xs-offset-4" src="https://static.igem.org/mediawiki/2017/a/a0/2017_HUST_China_model_equation6.png" />
−
                                    <li>①  Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.</li>
+
                    <p class="col-xs-12">We should also modify the recycling function. It will only use to sensed bacteria and captured bacteria. </p>
−
                                    <li>②  Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1ml of 1xPBS</li>
+
                    <p class="col-xs-12">Finally, the model is over. Running the model, the result is:</p>
−
                                    <li>③  Dilute the 2x fluorescein stock solution using 1xPBS to make a 1x fluorescein solution (final concentration is 50 μM).</li>
+
                    <section class="col-xs-6 col-xs-offset-1">
−
                                    <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>
+
                        <figure>
−
                                    <li>⑤Import data into "Abs600" blue cells in provided Excel calibration sheet</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">
−
                                </ul>
+
                                <source type="video/webm" src="https://static.igem.org/mediawiki/2017/6/60/2017_HUST_China_model_vedio3.2.webm" />
−
                            </li>
+
                                <track src="subs/TOS-arabic.srt" kind="subtitles" srclang="ar" label="Arabic" />
−
                     <h5>Measurement work flow：</h5>
+
                                <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>
 +
                     <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/f/f8/2017_HUST_China_interlab_img_001.png" rel="lightbox-demo" title="my caption"><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 />
−
                         <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">
+
                         <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">
 
                     </a>
 
                     </a>
−
                     <div class="col-xs-12" >
+
                     <p class="col-xs-12">Put these curve into one figure:</p>  
−
                        <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.
+
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                                </ul>
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                            </li>
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                    </div>
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                <div id="section6" 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;">Cell Culture Setup and Measurement</h3>
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                     <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/e/e6/2017_HUST_China_interlab_img_002.png" rel="lightbox-demo" title="my caption"><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 />
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                         <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">
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                         <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">
 
                     </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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                         <h5><strong>Transformation:</strong> </h5>
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                         <h4 style="color:#97477c"><strong>Result:</strong></h4>
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                            <ul class="yuandian">
+
                        <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>
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                                <li> control</li>
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                         <section class="col-xs-6 col-xs-offset-1">
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                                <li> Negative control</li>
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                            <figure>
−
                                <li>Test Device 1: J23101+I13504</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">
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                                <li>Test Device 2: J23106+I13504</li>
+
                                     <source type="video/webm" src="https://static.igem.org/mediawiki/2017/6/60/2017_HUST_China_model_vedio3.2.webm" />
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                                <li>Test Device 3: J23117+I13504</li>
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                                     <track src="subs/TOS-arabic.srt" kind="subtitles" srclang="ar" label="Arabic" />
−
                                <li>Test Device 4: J23101.BCD2.E0040.B0015</li>
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                                    <track src="subs/TOS-japanese.srt" kind="subtitles" srclang="jp" label="Japanese" />
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                                <li>Test Device 5: J23106.BCD2.E0040.B0015</li>
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                                    <track src="subs/TOS-english.srt" kind="subtitles" srclang="en" label="English" />
−
                                <li>Test Device 6: J23117.BCD2.E0040.B0015</li>
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                                     <track src="subs/TOS-turkish.srt" kind="subtitles" srclang="tr" label="Turkish" />
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                            </ul>
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                                     <track src="subs/TOS-ukrainian.srt" kind="subtitles" srclang="uk" label="Ukrainian" />
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                         <h5><strong>Cell growth:</strong> </h5>
+
                                 </video>
−
                        <li class="dropdown" style="list-style-type:none;">
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                             </figure>
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                                 <button href="#" class="dropdown-toggle" data-toggle="dropdown">
+
                         </section>
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                                    More details <b class="caret"></b>
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                                </button>
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                                <ul class="dropdown-menu" style="padding: 10px; background:#ccc;">
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                                     <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).
+
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                                    </a>
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−
                                     </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>
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                        <h5><strong>Cell growth, sampling, and assay</strong> </h5>
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                        <li class="dropdown" style="list-style-type:none;">
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                                <button href="#" class="dropdown-toggle" data-toggle="dropdown">
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                                    More details <b class="caret"></b>
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                                </button>
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                                <ul class="dropdown-menu" style="padding: 10px; background:#ccc;">
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+
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                                    <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>
+
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+
−
 
+
−
                                 </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">
−
                        <h5><strong>What type of media did you use for this step? </strong> </h5>
+
                         <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>
−
                         <p>Luria Bertani </p>
+
                         <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>
−
                        <h5><strong>What type of vessel or container did you use to grow your cells? </strong> </h5>
+
                         <p>In order to show you more details, we sum each areas’ concentration and put them into on figure:</p>
−
                         <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/2017/8/87/2017_HUST_China_interlab_img_003.png" rel="lightbox-demo" title="my caption"><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 />
−
                             <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">
+
                             <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">
 
                         </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>
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                <div id="section7" style="border: solid 1px #666; margin:5px; padding: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;">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/>
 
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                        <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">
 
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                    <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>
 
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                    <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/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>
 
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                    <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/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">
 
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                        </a>
 
−
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Revision as of 16:12, 28 October 2017

Model

「Model」

Overview

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.

Assumptions

The variability of temperature has no effect on our Reebot or it could be ignored.

Liquid in the container is stationary.

There is nothing else in the container which can influence Reebot.

Constant & Parameter

The description of constant, their values and the references involved in this model are listed in this table.

Constant Description
f1 concentration of Initially Bacteria
f2 concentration of Lanthanide ion
f3 concentration of Sensed Bacteria
f4 concentration of Captured Bacteria
f5 concentration of recycled sensed bacteria
f6 concentration of recycled captured bacteria

The description of parameter, their values and the references involved in this model are listed in the second table.

Parameter Description
dif the diffuse ability of bacteria
difl the diffuse ability of lanthanide ion
deadp dead/birth per minute of bacteria
vlcx velocity of movement on x label
vlcy velocity of movement on z label
vlcz concentration of recycled captured bacteria
krec speed of bacteria recycled on Si-board
kabcs speed of bacteria recycled on Si-board
rmax the amount of bacteria recyclyed on each area of Si-board
csen the lowest concentration lanthanide ion that can be sensed
ksen speed of bacteria sensing the lanthanide ion
kcap speed of sensed bacteria capturing the lanthanide ion
ccap the lowest concentration lanthanide ion that can be captured
LBlever The amount of lanthanide ion captured by one bacteria

Basical Diffusion

Rome was not built in a day. At the beginning, we built a basical diffusion model, just setting the diffusion function in our model.

The function is:

We release some Reebot to the top of our hypothetical container as initial value of f1, and use ‘ode’ tool in MATLAB to run this function. Here is the result:

Recycling

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.

Firstly, we should change the diffusion function:

(In the end of all function, the ∂f/∂t will be delta.)

And we set the area of Si-board, if it is in Si-board area:

Bacteria that combined with Si-board maybe abscess. So taking the abscission into consideration, we should add this function:

Finally:

Set a 5×5 Si-board on the top and then run the model, the result is:

Sensing and Capture

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 f1, concentration of lanthanide ion as f2, concentration of sensed bacteria（sensed the lanthanide ion but haven’t captured）as f3 and concentration of captured bacteria (have captured enough lanthanide ion) as f4.

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:

We should also modify the recycling function. It will only use to sensed bacteria and captured bacteria.

Finally, the model is over. Running the model, the result is:

For more intuitive, we draw a figure :

Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.



demo1

Put these curve into one figure:

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demo1

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.

Result:

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:

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.

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.

In order to show you more details, we sum each areas’ concentration and put them into on figure:

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demo1

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.

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.

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.

The video and figure is here.

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.

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.

Acknowledgments: