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

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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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#pic{
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<h1>InterLab</h1>
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<h3>Bronze Medal Criterion #4</h3>
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<p><b>Standard Tracks:</b> Participate in the Interlab Measurement Study and/or obtain new, high quality experimental characterization data for an existing BioBrick Part or Device and enter this information on that part's Main Page in the Registry. The part that you are characterizing must NOT be from a 2018 part number range.
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<br><br>
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For teams participating in the <a href="https://2018.igem.org/Measurement/InterLab">InterLab study</a>, all work must be shown on this page.
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        <h2 class="pageTitle">Interlab</h2>
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                  <div style="text-align: center">
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                  <h3><strong><span class="red-content">Introduction</span></strong></h3>
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                  </div>
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                    <p>
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                    This year, we HUST-China have volunteered to iGEM's fifth International InterLaboratory Measurement Study, in order to help the<br/>
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                    iGEM community collect data about how  reliable will  devices turn out to be in labs around the world.
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                  <img src="https://static.igem.org/mediawiki/2018/e/e9/T--HUST-China--2018-Intelab-pic14.png" class="img-responsive">
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                  </div>
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                  <li>
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                  <div class="collapsible-header"><i><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/09/T--HUST-China--2018-Intelab-pic11.png"></i>Provenance and release</div>
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                  <div class="collapsible-body">
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                    <div class="col-md-12 content-text" style="padding:10px;">
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                      <h3>①Individuals responsible for conducting InterLab study</h3>
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                    </div>
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                    <div class="col-md-10">
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                      <table class="table table-bordered table-hover" style="text-align: center;">
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                            <tr>
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                              <th>individuals</th>
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                              <th>Interlab parts</th>
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                            </tr>
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                            <tr>
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                              <td>Ziyang Xiao</td>
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                              <td>Created devices</td>
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                            </tr>
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                            <tr>
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                              <td>Ziyang Xiao, Haotian Ren, Yan Chen, Hao Qiu</td>
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                              <td> Conducted the measurements</td>
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                            </tr>
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                            <tr>
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                              <td>Ziyang Xiao</td>
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                              <td>Processed the data</td>
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                            </tr>
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                      </table>
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                  <div class="col-md-12 content-text" style="padding:10px;">
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                      <h3>②Corresponding email</h3>
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                  </div>
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                  <div class="col-md-10">
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                            <tr>
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                              <th>individuals</th>
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                              <th>emails</th>
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                            </tr>
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                            <tr>
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                              <td>Ziyang Xiao</td>
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                              <td>u201612166@hust.edu.cn</td>
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                            </tr>
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                            <tr>
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                              <td>Yan Chen</td>
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                              <td>u201713204@hust.edu.cn</td>
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                            </tr>
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                            <tr>
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                              <td> Haotian Ren</td>
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                              <td>u201712893@hust.edu.cn</td>
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                            </tr>
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                            <tr>
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                                <td>
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                                Hao  Qiu
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                                </td>
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                                <td>
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                                  u201713109@hust.edu.cn
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                                </td>
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                              </tr>
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                      </table>
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                </li>
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                <li>
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                  <div class="collapsible-header"><i><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/09/T--HUST-China--2018-Intelab-pic11.png"></i>Chassis and safety</div>
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                  <div class="collapsible-body">
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                  <p>
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                                  What chassis did you use?</br>
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                                  Escherichia coli DH5α</br>
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                                  What Biosafety Level is your chassis?</br>
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                                  BSL1</br>
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                                  What PPE did you utilize during your experiments?</br>
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                                  Tianming gloves</br>
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                                Songxinjiujiu labcoats</p>
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                  </div>
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                </li>
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                <li>
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                  <div class="collapsible-header"><i><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/09/T--HUST-China--2018-Intelab-pic11.png"></i>Instrument</div>
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                  <div class="collapsible-body" >
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                    <p>
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                            What instrument did you use during your measurements?</br>
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                              plate reader</br>
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 +
                        Please provide the brand and model of your instrument.</br>
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                          Flexstation 3</p>
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                  </div>
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                </li>
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                <li>
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                  <div class="collapsible-header"><i><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/09/T--HUST-China--2018-Intelab-pic11.png"></i>Calibration 1：OD600 reference point-LUDOX</div>
 +
                  <div class="collapsible-body">
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                      <div class="col-md-12 info-blocks">
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                          <i class="icon-info-blocks material-icons">photo_filter</i>
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                              <h3>a.Protocol</h3>
 +
                              <p> Use pathlength correction</br>
 +
                              Number of flashes per well：6</br>
 +
                              Orbital averaging (nm)：600</br>
 +
                              Temperature setting：22℃</br>
 +
                              Type of 96-well plate：Black plate (preferred)
 +
                              Our plates have flat-bottomed wells.</p>
 +
                          </div>
 +
                          </div>
 +
                     
 +
               
 +
                      <div class="col-md-12 info-blocks">
 +
                          <i class="icon-info-blocks material-icons">tune</i>
 +
                          <div class="info-blocks-in">
 +
                              <h3>b.Measurement Steps</h3>
 +
                              <p align="left">Add 100ul LUDOX into wells A1, B1, C1,D1</br>
 +
                              Add 100ul ddH2O into wells A2, B2, C2,D2</br>
 +
                                Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for
 +
                                cell measurements </br>
 +
                                Record the data in the table below or in your notebook</br>
 +
                                data into Excel sheet provided (OD600 reference point tab)</p>
 +
                          </div>
 +
                        </div>
 +
           
 +
                  <div class="col-md-10 col-md-offset-1" align="center"> 
 +
                <h4>Result</h4>
 +
                  <img src="https://static.igem.org/mediawiki/2018/b/bb/T--HUST-China--2018-Intelab-pic5.png" class="img-responsive">
 +
                    </div>
 +
                  </div>
 +
                </li>
 +
                <li>
 +
                  <div class="collapsible-header"><i><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/09/T--HUST-China--2018-Intelab-pic11.png"></i>Calibration 2：Particle standard curve-microsphere </div>
 +
                  <div class="collapsible-body" align="center">
 +
 +
 +
 +
                    <div class="col-md-12 info-blocks">
 +
                          <i class="icon-info-blocks material-icons">bubble_chart</i>
 +
                          <div class="info-blocks-in">
 +
                              <h3>a.Protocol</h3>
 +
                              <p align="left"> Use pathlength correction</br>
 +
                              Number of flashes per well：6</br>
 +
                              Orbital averaging (nm)：600</br>
 +
                              Temperature setting：22℃</br>
 +
                              Type of 96-well plate：Black plate (preferred)</br>
 +
                              Our plates have flat-bottomed wells.</p> 
 +
                          </div>
 +
                          </div>
 +
                          <div class="col-md-12 info-blocks">
 +
                          <i class="icon-info-blocks material-icons">star_half</i>
 +
                          <div class="info-blocks-in">
 +
                              <h3>b.Measurement Steps</h3>
 +
                              <p align="left"> Prepare the Microsphere Stock Solution: </br>
 +
                              ❏Obtain the tube labeled “Silica Beads” from the InterLab test kit
 +
                                and vortex</br>
 +
                              ❏vigorously for 30 seconds. </br>
 +
                              ❏Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube</br>
 +
                         
 +
                              ❏Add 904 μL of ddH2O to the microspheres </br>
 +
                              ❏Vortex well. This is your Microsphere Stock Solution.</p> 
 +
                          </div>
 +
                          </div>
 +
                     
 +
                                   
 +
               
 +
                  <div class="col-md-12">
 +
                  Prepare the serial dilution of Microspheres:
 +
                  <img src="https://static.igem.org/mediawiki/2018/d/db/T--HUST-China--2018-Intelab-pic7.png" class="img-responsive" ></div>
 +
 +
                    <div class="col-md-12 info-blocks">
 +
                          <i class="icon-info-blocks material-icons">polymer</i>
 +
                          <div class="info-blocks-in">
 +
                             
 +
                              <p align="left">❏Add 100 μl of ddH2O into wells A2, B2, C2, D2....A12, B12, C12, D12</br>
 +
                              ❏Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds</br>
 +
                              ❏Immediately add 200 μl of microspheres stock solution into A1 </br>
 +
                              ❏Transfer 100 μl of microsphere stock solution from A1 into A2. </br>
 +
                              ❏Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…</br>
 +
                              ❏Mix A3 by pipetting up and down 3x and transfer 100 μl into A4... </br>
 +
                              ❏Mix A4 by pipetting up and down 3x and transfer 100 μl into A5... </br>
 +
                              ❏Mix A5 by pipetting up and down 3x and transfer 100 μl into A6... </br>
 +
                              ❏Mix A6 by pipetting up and down 3x and transfer 100 μl into A7... </br>
 +
                              ❏Mix A7 by pipetting up and down 3x and transfer 100 μl into A8... </br>
 +
                              </p> 
 +
                          </div>
 +
                          </div>
 +
                     
 +
                        <div class="col-md-12 info-blocks">
 +
                          <i class="icon-info-blocks material-icons">spa</i>
 +
                          <div class="info-blocks-in">
 +
                             
 +
                              <p align="left">
 +
                              ❏ Mix A8 by pipetting up and down 3x and transfer 100 μl into A9... </br>
 +
                    ❏Mix A9 by pipetting up and down 3x and transfer 100 μl into A10... </br>
 +
                    ❏Mix A10 by pipetting up and down 3x and transfer 100 μl into A11... </br>
 +
                    ❏Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste</br>
 +
                    ❏ Repeat dilution series for rows B, C, D </br>
 +
                    ❏Re-Mix each row of your plate immediately before putting in the plate
 +
                    reader! Take care to mix gently and avoid creating bubbles on the surface of the liquid.</br>
 +
                    ❏ Measure Abs600 of all samples in instrument </br>
 +
                    ❏ Record the data in your notebook </br>
 +
                    ❏ Import data into Excel sheet provided (particle standard curve tab)
 +
                  </p>
 +
                          </div>
 +
                          </div>
 +
                      <div class="col-md-10 col-md-offset-1" align="center">
 +
     
 +
                    <h4>Result</h4>
 +
 +
                    <img src="https://static.igem.org/mediawiki/2018/1/1a/T--HUST-China--2018-Intelab-pic31.png" class="img-responsive" >
 +
                    <img src="https://static.igem.org/mediawiki/2018/archive/e/ec/20181013122210%21T--HUST-China--2018-Intelab-pic30.png" class="img-responsive" >
 +
                          </div>
 +
                  </div>
 +
                </li>
 +
                <li>
 +
                  <div class="collapsible-header"><i><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/09/T--HUST-China--2018-Intelab-pic11.png"></i>Calibration 3：Fluorescence standard curve – Fluorescein
 +
                  </div>
 +
                  <div class="collapsible-body" align="center">
 +
 +
 +
                                <div class="col-md-12 info-blocks">
 +
                          <i class="icon-info-blocks material-icons">grain</i>
 +
                          <div class="info-blocks-in">
 +
                            <h3>a.Protocol</h3>
 +
                              <p align="left">
 +
                              Use pathlength correction</br>
 +
                    Number of flashes per well：
 +
                &emsp;6</br>
 +
                    Gain setting:
 +
                &emsp;Automatic</br>
 +
                  It passed 530nm when we used a filter.</br>
 +
                    Emission wavelength:
 +
                &emsp; 525nm</br>
 +
                    Excitation wavelength:
 +
                  &emsp; 485nm</br>
 +
                    Fluorescence reading:
 +
                  &emsp; Bottom optic</br>
 +
                    Type of 96-well plate：
 +
                  &emsp; Black plate (preferred)</br>
 +
                    Our plates have flat-bottomed wells.</br>
 +
                    Temperature setting:
 +
                  &emsp; 22℃</br>
 +
                  </p>
 +
                          </div>
 +
                          </div>
 +
 +
 +
                                <div class="col-md-12 info-blocks">
 +
                          <i class="icon-info-blocks material-icons">fingerprint</i>
 +
                          <div class="info-blocks-in">
 +
                            <h3>b.Measurement Steps</h3>
 +
                              <p align="left">
 +
                      Prepare the fluorescein stock solution: </br>
 +
                  ❏ Spin down fluorescein kit tube to make sure pellet is at the bottom of
 +
                      tube.</br>
 +
                  ❏ Prepare 10x fluorescein stock solution (100 μM) by resuspending</br>
 +
                    fluorescein in 1 mL of 1xPBS.
 +
                  ❏ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x</br>
 +
                    fluorescein solution with concentration 10 μM: 100 μL of 10x fluorescein</br>
 +
                    stock into 900 μL 1x PBS</br></br>
 +
                  </p>
 +
                          </div>
 +
                          </div>
 +
 +
 +
                   
 +
                  <p> <h3> Prepare the serial dilutions of fluorescein:</h3></p>
 +
                    <img src="https://static.igem.org/mediawiki/2018/d/db/T--HUST-China--2018-Intelab-pic7.png" class="img-responsive" >
 +
 +
                            <div class="col-md-12 info-blocks">
 +
                          <i class="icon-info-blocks material-icons">flare</i>
 +
                          <div class="info-blocks-in">
 +
                         
 +
                              <p align="left">
 +
                        ❏Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12 </br>
 +
                        ❏ Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1</br>
 +
                        ❏ Transfer 100 μl of fluorescein stock solution from A1 into A2.</br>
 +
                        ❏ Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…</br>
 +
                        ❏ Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...</br>
 +
                        ❏Mix A4 by pipetting up and down 3x and transfer 100 μl into A5... </br>
 +
                        ❏ Mix A5 by pipetting up and down 3x and transfer 100 μl into A6... </br>
 +
                        ❏ Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...
 +
                  </p>
 +
                          </div>
 +
                          </div>
 +
 +
                          <div class="col-md-12 info-blocks">
 +
                          <i class="icon-info-blocks material-icons">extension</i>
 +
                          <div class="info-blocks-in">
 +
                         
 +
                              <p align="left">
 +
                       
 +
                        ❏ Mix A7 by pipetting up and down 3x and transfer 100 μl into A8... </br>
 +
                        ❏ Mix A8 by pipetting up and down 3x and transfer 100 μl into A9... </br>
 +
                        ❏ Mix A9 by pipetting up and down 3x and transfer 100 μl into A10... </br>
 +
                        ❏ Mix A10 by pipetting up and down 3x and transfer 100 μl into A11... </br>
 +
                        ❏ Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid
 +
                        waste</br>
 +
                        ❏ Repeat dilution series for rows B, C, D </br>
 +
                        ❏ Measure fluorescence of all samples in instrument</br>
 +
                        ❏ Record the data in your notebook </br>
 +
                        ❏ Import data into Excel sheet provided(fluorescein standard curve tab)
 +
                     
 +
                  </p>
 +
                          </div>
 +
                          </div>
 +
                           
 +
                       
 +
                 
 +
                   
 +
                      <div div class="col-md-10 col-md-offset-1" align="center">
 +
                        <h4>Result</h4>
 +
                        <img src="https://static.igem.org/mediawiki/2018/e/ec/T--HUST-China--2018-Intelab-pic30.png" class="img-responsive" >
 +
                        <img src="https://static.igem.org/mediawiki/2018/2/24/T--HUST-China--2018-Intelab-pic33.png" class="img-responsive" >
 +
                        <img src="https://static.igem.org/mediawiki/2018/3/34/T--HUST-China--2018-Intelab-pic34.png" class="img-responsive" >
 +
 +
 +
                  </div>
 +
                </li>
 +
                <li>
 +
                  <div class="collapsible-header"><i><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/09/T--HUST-China--2018-Intelab-pic11.png"></i>Cell culture setup and meaturement</div>
 +
                  <div class="collapsible-body" align="center">
 +
                      <img src="https://static.igem.org/mediawiki/2018/3/34/T--HUST-China--2018-Intelab-pic34.png" class="img-responsive" >
 +
                         
 +
                            <div class="col-md-12 info-blocks">
 +
                          <i class="icon-info-blocks material-icons">all_inclusive</i>
 +
                          <div class="info-blocks-in">
 +
                             
 +
                              <p align="left">
 +
                              Transformation:</br>
 +
                          Negative control BBa_R0040 Kit Plate 7 Well 2D</br>
 +
                          Positive control BBa_I20270 Kit Plate 7 Well 2B </br>
 +
                          Test Device 1 BBa_J364000 Kit Plate 7 Well 2F</br>
 +
                          Test Device 2 BBa_J364001 Kit Plate 7 Well 2H </br>
 +
                          Test Device 3 BBa_J364002 Kit Plate 7 Well 2J </br>
 +
                          Test Device 4 BBa_J364007 Kit Plate 7 Well 2L </br>
 +
                          Test Device 5 BBa_J364008 Kit Plate 7 Well 2N </br>
 +
                          Test Device 6 BBa_J364009 Kit Plate 7 Well 2P </p>
 +
                        </br>
 +
                          <p align="left">Cell grow:</br>
 +
                          Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm. </p>
 +
                          </div>
 +
                          </div>
 +
 +
 +
                          <div class="col-md-12 info-blocks">
 +
                          <i class="icon-info-blocks material-icons">fingerprint</i>
 +
                          <div class="info-blocks-in">
 +
                             
 +
                              <p  align="left">
 +
                            Cell growth, sampling, and assay</br>
 +
                           Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of
 +
                          culture into 4.5mL of LB+Chlor)</br>
 +
                           Measure Abs600 of these 1:10 diluted cultures</br>
 +
                         Record the data in your notebook </br>
 +
                         Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB
 +
                                      medium + Chloramphenicol in 50 mL falcon tube</br>
 +
                            Take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes,
 +
                                        prior to incubation. Place the samples on ice. </br>
 +
                            Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.</br>
 +
                          Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf
 +
                          tubes. Place samples on ice. </br>
 +
                           At the end of sampling point you need to measure your samples (Abs600 and
 +
                          fluorescence measurement) </br>
 +
                            Record data in  notebook </br>
 +
                           Import data into Excel sheet provided (fluorescence measurement tab)
 +
                          </p>
 +
                          </div>
 +
                          </div>
 +
 +
 +
     
 +
                         
 +
                        <!-- 表格-->
 +
                              <div class="col-md-10 col-md-offset-1">
 +
                      <table class="table table-bordered table-hover" style="text-align: center;">
 +
                          <p><h3>The initial OD600 measurement of our overnight cultures</h3></p>
 +
                            <tr>
 +
                              <th>Sample</th>
 +
                              <th>Abs600 reading replicate1</th>
 +
                              <th>Abs600 reading replicate2</th>
 +
                            </tr>
 +
                            <tr>
 +
                              <td>Negative control</td>
 +
                              <td>0.106</td>
 +
                              <td>0.104</td>
 +
                            </tr>
 +
                              <tr>
 +
                              <td>Positive control</td>
 +
                              <td>0.107</td>
 +
                              <td>0.108</td>
 +
                            </tr>
 +
                            <tr>
 +
                              <td>Device1</td>
 +
                              <td>0.0937</td>
 +
                              <td>0.108</td>
 +
                            </tr>
 +
                            <tr>
 +
                              <td>Device2</td>
 +
                              <td>0.119</td>
 +
                              <td>0.105</td>
 +
                            </tr>
 +
                            <tr>
 +
                              <td>Device3</td>
 +
                              <td>0.106</td>
 +
                              <td>0.116</td>
 +
                            </tr>
 +
                            <tr>
 +
                              <td>Device4</td>
 +
                              <td> 0.109</td>
 +
                              <td>0.104</td>
 +
                            </tr>
 +
                            <tr>
 +
                              <td>Device5</td>
 +
                              <td>0.106</td>
 +
                              <td>0.104</td>
 +
                            </tr>
 +
                            <tr>
 +
                              <td>Device6</td>
 +
                              <td>0.104</td>
 +
                              <td>0.109</td>
 +
                            </tr>
 +
                            <tr>
 +
                              <td>Negative control</td>
 +
                              <td>0.106</td>
 +
                              <td>0.104</td>
 +
                            </tr>
 +
                      </table>
 +
                  </div>
 +
 +
 +
 +
                   
 +
                  <!-- 表格-->
 +
 +
                            <div class="col-md-12 info-blocks">
 +
                          <i class="icon-info-blocks material-icons">hdr_weak</i>
 +
                          <div class="info-blocks-in">
 +
                             
 +
                              <p align="left">
 +
                               Type of media we used for this step: Luria Bertani<br/>
 +
                         Type of vessel or container we used to grow cells: 50 ml Falcon tube<br/>
 +
                         Temperature setting:22℃<br/>
 +
                         Type of 96-well plates: Black plates with transparent/clear bottom (preferred),flat<br/>
 +
                         Measurement: Measure OD and fluorescence of all samples<br/>
 +
                           Suggested Plate Layout for 96-well Plate<br/>
 +
                          </p>
 +
                          </div>
 +
                          </div>
 +
 +
 +
 +
                          <img src="https://static.igem.org/mediawiki/2018/archive/7/78/20181013124643%21T--HUST-China--2018-Intelab-final.png" class="img-responsive" >
 +
                          <h3> Result</h3>
 +
                          <img src="https://static.igem.org/mediawiki/2018/archive/7/78/20181013124711%21T--HUST-China--2018-Intelab-final.png" class="img-responsive" >
 +
                          <img src="https://static.igem.org/mediawiki/2018/archive/7/78/20181013124733%21T--HUST-China--2018-Intelab-final.png" class="img-responsive" >
 +
                          <img src="https://static.igem.org/mediawiki/2018/archive/7/78/20181013124735%21T--HUST-China--2018-Intelab-final.png" class="img-responsive" >
 +
                          <img src="https://static.igem.org/mediawiki/2018/archive/7/78/20181013124826%21T--HUST-China--2018-Intelab-final.png" class="img-responsive" >
 +
                          <img src="https://static.igem.org/mediawiki/2018/archive/7/78/20181013124901%21T--HUST-China--2018-Intelab-final.png" class="img-responsive" >
 +
                          <img src="https://static.igem.org/mediawiki/2018/archive/7/78/20181013125039%21T--HUST-China--2018-Intelab-final.png" class="img-responsive" >
 +
                          <img src="https://static.igem.org/mediawiki/2018/archive/7/78/20181013125106%21T--HUST-China--2018-Intelab-final.png" class="img-responsive" >
 +
                          <img src="https://static.igem.org/mediawiki/2018/7/78/T--HUST-China--2018-Intelab-final.png" class="img-responsive" >
 +
                         
 +
                             
 +
                  </div>
 +
                </li>
 +
               
 +
                  <li>
 +
                  <div class="collapsible-header"><i><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/09/T--HUST-China--2018-Intelab-pic11.png"></i>Counting colony-forming units (CFUs) from the sample.</div>
 +
                  <div class="collapsible-body">
 +
                            <div class="col-md-10 col-md-offset-1">
 +
                       
 +
                          <p align="left">This measurement, related to cell concentration of the culture, can be used to calibrate OD600 to colony forming unit (CFU) counts.<br/>
 +
                          We measured and dilute our overnight cultures to OD600=0.1, and we checked the OD600 to ensure we have diluted to 0.1.Also,we did this in triplicate for each culture.
 +
                            </p>
 +
                          </div>
 +
                              <div class="col-md-10 col-md-offset-1">
 +
                            <img src="https://static.igem.org/mediawiki/2018/e/e8/T--HUST-China--2018-Intelab-final11.png" class="img-responsive" >
 +
                          </div>
 +
         
 +
                </li>
 +
              </div>
 +
              </ul>
 +
 +
 +
                          <!-- Accordion ends -->
 +
             
 +
</div>
 +
 +
            </div>
 +
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        </div>           
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Latest revision as of 23:02, 17 October 2018

HillSide Multi purpose HTML5 Template

Interlab

Introduction

This year, we HUST-China have volunteered to iGEM's fifth International InterLaboratory Measurement Study, in order to help the
iGEM community collect data about how reliable will devices turn out to be in labs around the world.

  • Provenance and release

    ①Individuals responsible for conducting InterLab study

    individuals Interlab parts
    Ziyang Xiao Created devices
    Ziyang Xiao, Haotian Ren, Yan Chen, Hao Qiu Conducted the measurements
    Ziyang Xiao Processed the data

    ②Corresponding email

    individuals emails
    Ziyang Xiao u201612166@hust.edu.cn
    Yan Chen u201713204@hust.edu.cn
    Haotian Ren u201712893@hust.edu.cn
    Hao Qiu u201713109@hust.edu.cn
  • Chassis and safety

    What chassis did you use?
    Escherichia coli DH5α
    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 1：OD600 reference point-LUDOX
    photo_filter

    a.Protocol

    Use pathlength correction
    Number of flashes per well：6
    Orbital averaging (nm)：600
    Temperature setting：22℃
    Type of 96-well plate：Black plate (preferred) Our plates have flat-bottomed wells.

    tune

    b.Measurement Steps

    Add 100ul LUDOX into wells A1, B1, C1,D1
    Add 100ul ddH2O into wells A2, B2, C2,D2
    Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements
    Record the data in the table below or in your notebook
    data into Excel sheet provided (OD600 reference point tab)

    Result

  • Calibration 2：Particle standard curve-microsphere
    bubble_chart

    a.Protocol

    Use pathlength correction
    Number of flashes per well：6
    Orbital averaging (nm)：600
    Temperature setting：22℃
    Type of 96-well plate：Black plate (preferred)
    Our plates have flat-bottomed wells.

    star_half

    b.Measurement Steps

    Prepare the Microsphere Stock Solution:
    ❏Obtain the tube labeled “Silica Beads” from the InterLab test kit and vortex
    ❏vigorously for 30 seconds.
    ❏Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube
    ❏Add 904 μL of ddH2O to the microspheres
    ❏Vortex well. This is your Microsphere Stock Solution.

    Prepare the serial dilution of Microspheres:
    polymer

    ❏Add 100 μl of ddH2O into wells A2, B2, C2, D2....A12, B12, C12, D12
    ❏Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds
    ❏Immediately add 200 μl of microspheres stock solution into A1
    ❏Transfer 100 μl of microsphere stock solution from A1 into A2.
    ❏Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…
    ❏Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...
    ❏Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
    ❏Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...
    ❏Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...
    ❏Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...

    spa

    ❏ Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...
    ❏Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...
    ❏Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...
    ❏Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste
    ❏ Repeat dilution series for rows B, C, D
    ❏Re-Mix each row of your plate immediately before putting in the plate reader! Take care to mix gently and avoid creating bubbles on the surface of the liquid.
    ❏ Measure Abs600 of all samples in instrument
    ❏ Record the data in your notebook
    ❏ Import data into Excel sheet provided (particle standard curve tab)

    Result

  • Calibration 3：Fluorescence standard curve – Fluorescein
    grain

    a.Protocol

    Use pathlength correction
    Number of flashes per well：  6
    Gain setting:  Automatic
    It passed 530nm when we used a filter.
    Emission wavelength:   525nm
    Excitation wavelength:   485nm
    Fluorescence reading:   Bottom optic
    Type of 96-well plate：   Black plate (preferred)
    Our plates have flat-bottomed wells.
    Temperature setting:   22℃

    fingerprint

    b.Measurement Steps

    Prepare the fluorescein stock solution:
    ❏ Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
    ❏ Prepare 10x fluorescein stock solution (100 μM) by resuspending
    fluorescein in 1 mL of 1xPBS. ❏ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x
    fluorescein solution with concentration 10 μM: 100 μL of 10x fluorescein
    stock into 900 μL 1x PBS

    Prepare the serial dilutions of fluorescein:

    flare

    ❏Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
    ❏ Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1
    ❏ Transfer 100 μl of fluorescein stock solution from A1 into A2.
    ❏ Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…
    ❏ Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...
    ❏Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
    ❏ Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...
    ❏ Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...

    extension

    ❏ Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...
    ❏ Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...
    ❏ Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...
    ❏ Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...
    ❏ Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste
    ❏ Repeat dilution series for rows B, C, D
    ❏ Measure fluorescence of all samples in instrument
    ❏ Record the data in your notebook
    ❏ Import data into Excel sheet provided(fluorescein standard curve tab)

    Result

  • Cell culture setup and meaturement
    all_inclusive

    Transformation:
    Negative control BBa_R0040 Kit Plate 7 Well 2D
    Positive control BBa_I20270 Kit Plate 7 Well 2B
    Test Device 1 BBa_J364000 Kit Plate 7 Well 2F
    Test Device 2 BBa_J364001 Kit Plate 7 Well 2H
    Test Device 3 BBa_J364002 Kit Plate 7 Well 2J
    Test Device 4 BBa_J364007 Kit Plate 7 Well 2L
    Test Device 5 BBa_J364008 Kit Plate 7 Well 2N
    Test Device 6 BBa_J364009 Kit Plate 7 Well 2P


    Cell grow:
    Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.

    fingerprint

    Cell growth, sampling, and assay
     Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
     Measure Abs600 of these 1:10 diluted cultures
     Record the data in your notebook
     Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube
     Take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. Place the samples on ice.
     Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.
     Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice.
     At the end of sampling point you need to measure your samples (Abs600 and fluorescence measurement)
     Record data in notebook
     Import data into Excel sheet provided (fluorescence measurement tab)

    The initial OD600 measurement of our overnight cultures

    Sample Abs600 reading replicate1 Abs600 reading replicate2
    Negative control 0.106 0.104
    Positive control 0.107 0.108
    Device1 0.0937 0.108
    Device2 0.119 0.105
    Device3 0.106 0.116
    Device4 0.109 0.104
    Device5 0.106 0.104
    Device6 0.104 0.109
    Negative control 0.106 0.104
    hdr_weak

     Type of media we used for this step: Luria Bertani
     Type of vessel or container we used to grow cells: 50 ml Falcon tube
     Temperature setting:22℃
     Type of 96-well plates: Black plates with transparent/clear bottom (preferred),flat
     Measurement: Measure OD and fluorescence of all samples
     Suggested Plate Layout for 96-well Plate

    Result

  • Counting colony-forming units (CFUs) from the sample.

    This measurement, related to cell concentration of the culture, can be used to calibrate OD600 to colony forming unit (CFU) counts.
    We measured and dilute our overnight cultures to OD600=0.1, and we checked the OD600 to ensure we have diluted to 0.1.Also,we did this in triplicate for each culture.

College of Life Science & Technology
Huazhong University of Science and Technology
Add: 1037 Luoyu Road, Wuhan, Hubei, China; P.C: 430074
Copyright ©2018 Huazhong University of Science & Technology. Produced By HUST-China