Difference between revisions of "Team:Imperial College/Model"

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     <button class="collapsible" type="button">Electrochemical Model</button>
 
     <button class="collapsible" type="button">Electrochemical Model</button>
 
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</head>
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<body>
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<div class="container">         
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        <div class="content">
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            <div class="titleimg">
 +
                    <h1>Methods</h1>
 +
                    <br/>
 +
                    <br/>
 +
            <img src="https://static.igem.org/mediawiki/2018/7/71/T--Imperial_College--experiment.png" alt="" width="28%"; >
 +
            </div>
 +
 +
                <h3>Contents</h3>
 +
       
 +
            <div class="contents">
 +
                <ul>
 +
                    <li><a href="#expt1">Assembling the Pixcell Constructs</a></li>
 +
                    <li><a href="#expt2">Characterizing Pixcell Constructs</a></li>
 +
                    <li><a href="#expt3">Electrochemistry of Redox Modulators</a></li>
 +
                    <li><a href="#expt4">Spatial Electronic Control of Gene Induction</a></li>
 +
                    <li><a href="#expt5">Creating the Sox Library</a></li>
 +
                    <li><a href="#expt6">Characterising the Sox Library</a></li>
 +
                    <li><a href="#expt7">Testing Phenazine Methosulfate</a></li>
 +
                    <li><a href="#expt8">Biocontainment- Gp2 Growth Inhibition</a></li>
 +
                    <li><a href="#expt9">Fabric Bioprinting - Melanin</a></li>
 +
 +
                </ul>
 +
            </div>
 +
 +
               
 +
 +
                <h3 class="marginbottom">Experiments</h3>
 +
 +
         
 +
                <!--Start Experiment 1-->
 +
                <div id="expt1"></div>
 +
                <h4 class="underbold"> Assembling the Pixcell Constructs</h4>
 +
                <div onclick="expandAll('expt1p');" class="expandButton">
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                        <p class="expand expt1p colorChange" style="font-size:25px;padding-top:1px;" >Access All</p>
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                    <div class="expand" style="margin-right:5px;">
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                          <i class="fa fa-angle-double-down expt1p"></i>
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                    </div>
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                </div>
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                <div class="clr"></div>
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 +
                <div type="button" class="accordion"> Description </div>
 +
                <div class="description">
 +
                    <p>Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.</p>
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                </div>
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 +
                <div type="button" class="accordion">Materials</div>
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                <div class="panel expt1p">
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                    <p >Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.</p>
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                </div>
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                <div type="button" class="accordion">Notes</div>
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                <div class="panel expt1p">
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                        <p>Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
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                        Lorem ipsum dolor, sit amet consectetur adipisicing elit. <a href="#" class="highlight">Competant cell protocol</a> Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.</p>
 +
                        <p>Lorem ipsum dolor, sit amet <a href="#" class="highlight">Heatshock protocol</a> consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.</p>               
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                </div>
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 +
               
 +
                   
 +
                <div type="button" class="accordion" >Procedure</div>
 +
                <div class="panel expt1p">
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                    <p>Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.</p>
 +
                </div>
 +
                <div class="clr"></div>
 +
            <!--End Experiment-->
 +
 +
       
 +
                <!--Start Experiment 2-->
 +
                <div id="expt2">
 +
                <h4 class="underbold"> Characterizing Pixcell Constructs</h4>
 +
                <div onclick="expandAll('expt2p');" class="expandButton">
 +
                    <p class="expand expt2p colorChange" style="font-size:25px;padding-top:1px;" >Access All</p>
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                    <div class="expand" style="margin-right:5px;">
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                        <i class="fa fa-angle-double-down expt2p"></i>
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                    </div>
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                </div>
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                </div>
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                <div class="clr"></div>
 +
               
 +
                <div type="button" class="accordion"> Description </div>
 +
                <div class="description">
 +
                    <p>This protocol describes how to measure E. coli growth and GFP expression over time with a plate reader, using the optical density at 600 nm as signal for cell density and excitation and emission wavelengths of 475.
 +
All characterization was performed using the FLUOstar BMG Labtech in greiner bioone F-bottom dark plates with clear bottom, to decrease fluorescence background.
 +
Each cycle of this experiment takes two days and has to be repeated two days, in order to have 2 biological and 2 technical replicates, for a total of 4 days and 4 96-well plates used.
 +
</p>
 +
                </div>
 +
           
 +
                <div type="button" class="accordion">Materials</div>
 +
                <div class="panel expt2p">
 +
                  <ul>
 +
                            <li>Autoclaved LB media</li>
 +
                            <li>Autoclaved 10x concentated LB media</li>
 +
                            <li>500 uM pyocyanin stock solution (from Sigma-Aldrich, CAS: No. 86-66-5)</li>
 +
                            <li>1250 mM potassium ferricyanide stock solution (from Sigma-Aldrich, CAS: No. 13746-66-2)</li>                           
 +
                            <li>500 mM ferrocyanide stock solution (from Sigma-Aldrich, CAS 14459-95-1 )</li>
 +
                            <li>2% Sodium Sulfite Solution</li>
 +
                            <li>
 +
Agar plates with colonies of E. coli DJ901 strains with respective constructs: PixCell Patterning Circuit 1 (BBa_K2862021). PixCell Patterning Circuit 2 (BBa_K2862022), negative control (DJ901 WT) and positive control (constitutively expressed GFP).</li>
 +
                            <li>37°C shaking incubator</li>
 +
                            <li>BMG Labtech FLUOstar plate reader</li>
 +
                            <li>4x Greiner BioOne 96-F 96-well microplate (black with clear bottom)</li>
 +
                            <li>14 mL culture tubes</li>
 +
                            <li>autoclaved eppendorfs</li>
 +
                        </ul>
 +
                </div>
 +
 +
                <div type="button" class="accordion">Notes</div>
 +
                <div class="panel expt2p">
 +
                        <p>Work in very sterile conditions. To prevent contaminations in the blanks, use triple antibiotics.
 +
</br>
 +
Store pyocyanin and ferrocyanide and ferricyanide solutions in the freezer until use.
 +
</br>
 +
We used 10x concentrated LB in order not to dilute nutrients at higher pyocyanin concentration; this was back diluted with autoclaved water. Mastemixes have double the working concentrations, as 100 uL of solution is then diluted with 100 uL of liquid cultures.
 +
 +
                        </p>               
 +
                </div>
 +
               
 +
                  <div type="button" class="accordion" >Procedure</div>
 +
                <div class="panel expt2p">
 +
                    <p>Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.</p>
 +
<ol>
 +
<li>Grow starter liquid cultures of cells  (Day 0, 10-15 min preparation +  overnight)</li>
 +
 +
<p>Working in very sterile conditions, take 5 14mL culture tubes and prepare them according to table below: </p>
 +
 +
<table class="tg">
 +
  <tr>
 +
    <th class="tg-1wig">Label</th>
 +
    <th class="tg-1wig"><span style="font-style:italic">E. coli</span> strain</th>
 +
    <th class="tg-1wig">Construct</th>
 +
    <th class="tg-1wig">Antibiotic  (Ab)</th>
 +
    <th class="tg-1wig">V Ab (uL)</th>
 +
    <th class="tg-1wig">V LB (mL)</th>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">PC1</td>
 +
    <td class="tg-0lax">DJ901</td>
 +
    <td class="tg-0lax">BBa_K2862021</td>
 +
    <td class="tg-096r">Kan + Amp</td>
 +
    <td class="tg-0lax">5 + 5</td>
 +
    <td class="tg-0lax">5</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">PC2</td>
 +
    <td class="tg-0lax">DJ901</td>
 +
    <td class="tg-0lax">BBa_K2862022</td>
 +
    <td class="tg-0lax">Kan + Amp</td>
 +
    <td class="tg-0lax">5 + 5</td>
 +
    <td class="tg-0lax">5</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">-</td>
 +
    <td class="tg-0lax">DJ901</td>
 +
    <td class="tg-0lax">none</td>
 +
    <td class="tg-0lax">Kan</td>
 +
    <td class="tg-0lax">5 </td>
 +
    <td class="tg-0lax">5</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">+</td>
 +
    <td class="tg-0lax">DJ901</td>
 +
    <td class="tg-0lax">GFP_Boo</td>
 +
    <td class="tg-0lax">Kan + Chl</td>
 +
    <td class="tg-0lax">5 + 5</td>
 +
    <td class="tg-0lax">5</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">Blanks</td>
 +
    <td class="tg-0lax">None</td>
 +
    <td class="tg-0lax">None</td>
 +
    <td class="tg-0lax">Kan + Amp + Chl</td>
 +
    <td class="tg-0lax">5 + 5</td>
 +
    <td class="tg-0lax">5</td>
 +
  </tr>
 +
</table>
 +
<p>With a pipette tip pick a desired colony* from an agar plate and release the contaminated tip in the culture tip.  Place inoculated culture tubes in a 37° C in a shaking incubator overnight (for faster growth the angle between the vertical axis of the tube and the shaking plane of the incubator should be of 45°)</p>
 +
 +
<li>Prepare mastermixes (30 min)</li>
 +
<p>Mastermixes for each redox molecule concentration are prepared in 1.5 mL eppendorfs. The mastermixes are enough to fill 2 96-well microplates with 100 uL of solutions in each well, in order to have two replicate of the same experiment. Take 8 autoclaved 1.5 mL eppendorfs and prepare them as described in the table below.</p>
 +
<p>Mastermizes for Day 1 and Day 2 :</p>
 +
<table class="tg">
 +
  <tr>
 +
    <th class="tg-s268">Condition</th>
 +
    <th class="tg-s268">V Pyo (uL)</th>
 +
    <th class="tg-s268">Kan (uL)</th>
 +
    <th class="tg-0lax">LB 10x uL</th>
 +
    <th class="tg-0lax">Autoclaved Water (uL)</th>
 +
    <th class="tg-0lax">Tot Vol (uL)</th>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-s268">A = 0 uM Pyo</td>
 +
    <td class="tg-s268">0</td>
 +
    <td class="tg-s268">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">1098</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-s268">B = 0.01 uM Pyo</td>
 +
    <td class="tg-s268">0.0976</td>
 +
    <td class="tg-s268">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">1097.9024</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">C = 0.025 uM Pyo</td>
 +
    <td class="tg-0lax">0.244</td>
 +
    <td class="tg-0lax">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">1097.756</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">D = 0.05 uM Pyo</td>
 +
    <td class="tg-0lax">0.488</td>
 +
    <td class="tg-0lax">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">1097.512</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">E = 0.1 uM Pyo</td>
 +
    <td class="tg-0lax">0.976</td>
 +
    <td class="tg-0lax">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">1097.024</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">F = 0.25 uM Pyo</td>
 +
    <td class="tg-0lax">2.44</td>
 +
    <td class="tg-0lax">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">1095.56</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">G = 0.5 uM Pyo</td>
 +
    <td class="tg-0lax">4.88</td>
 +
    <td class="tg-0lax">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">1093.12</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">H = 1 uM Pyo</td>
 +
    <td class="tg-0lax">9.76</td>
 +
    <td class="tg-0lax">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">1088.24</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
</table>
 +
<p>Mastermixes for Day 3 and Day 4: </p>
 +
 +
<table class="tg">
 +
  <tr>
 +
    <th class="tg-s268">Condition</th>
 +
    <th class="tg-s268">V Pyo (uL)</th>
 +
    <th class="tg-s268">Kan (uL)</th>
 +
    <th class="tg-0lax">LB 10x uL</th>
 +
    <th class="tg-0lax">Autoclaved Water (uL)</th>
 +
    <th class="tg-0lax">Tot Vol (uL)</th>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-s268">A = 2.5 uM Pyo</td>
 +
    <td class="tg-s268">24.4</td>
 +
    <td class="tg-s268">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">1073.6</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-s268">B = 5 uM Pyo</td>
 +
    <td class="tg-s268">48.8</td>
 +
    <td class="tg-s268">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">1049.2</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">C = 10 uM Pyo</td>
 +
    <td class="tg-0lax">97.6</td>
 +
    <td class="tg-0lax">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">1000.4</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">D = 25 uM Pyo</td>
 +
    <td class="tg-0lax">244</td>
 +
    <td class="tg-0lax">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">854</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">E = 50 uM Pyo</td>
 +
    <td class="tg-0lax">488</td>
 +
    <td class="tg-0lax">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">610</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">F = 100 uM Pyo</td>
 +
    <td class="tg-0lax">976</td>
 +
    <td class="tg-0lax">2.44</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">122</td>
 +
    <td class="tg-0lax">1220</td>
 +
  </tr>
 +
</table>
 +
 +
<li>Fill 96-well plate with media solutions</li>
 +
<p>Add 100 uL of media in the desired wells, according following the label on mastermixes and table below: </p>
 +
<table class="tg">
 +
  <tr>
 +
    <th class="tg-0pky"></th>
 +
    <th class="tg-fymr">1</th>
 +
    <th class="tg-fymr">2</th>
 +
    <th class="tg-fymr">3</th>
 +
    <th class="tg-fymr">4</th>
 +
    <th class="tg-fymr">5</th>
 +
    <th class="tg-fymr">6</th>
 +
    <th class="tg-fymr">7</th>
 +
    <th class="tg-fymr">8</th>
 +
    <th class="tg-fymr">9</th>
 +
    <th class="tg-fymr">10</th>
 +
    <th class="tg-fymr">11</th>
 +
    <th class="tg-fymr">12</th>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-fymr">A</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-0pky">BA</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-0pky">BA</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-0pky">BA</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-0pky">BA</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-fymr">B</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-0pky">BB</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-0pky">BB</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-0pky">BB</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-0pky">BB</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-fymr">C</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-0pky">BC</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-0pky">BC</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-0pky">BC</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-0pky">BC</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-fymr">D</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-0pky">BD</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-0pky">BD</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-0pky">BD</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-0pky">BD</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-fymr">E</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-0pky">BE</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-0pky">BE</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-0pky">BE</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-0pky">BE</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-fymr">F</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-0pky">BF</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-0pky">BF</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-0pky">BF</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-0pky">BF</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-fymr">G</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-0pky">BG</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-0pky">BG</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-0pky">BG</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-0pky">BG</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-fymr">H</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-cai3">PC1</td>
 +
    <td class="tg-0pky">BH</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-f14n">PC2</td>
 +
    <td class="tg-0pky">BH</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-xuin">-</td>
 +
    <td class="tg-0pky">BH</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-r3a5">+</td>
 +
    <td class="tg-0pky">BH</td>
 +
  </tr>
 +
</table>
 +
<li>OD600 matching</li>
 +
<p>Once the cultures tubes in the shaking incubator appear cloudy, take them out and in a fresh microplate add 100 uL of each sample into a well. Add 100 uL of LB + antibiotic into a blank well. Take endpoint measurement of the filled wells and blank them with LB+antibiotic. Note down the OD600 value of the wells and dilute them to the desired initial OD600 (a value of 0.1 is suggested for a volume of 100 uL</p>
 +
<p>Cell dilution calculator:
 +
Volume of LB+Antibiotic to add in culture tube = [( Vcells * ODcell)/desired OD] - Vcells
 +
E.g. to dilute 5mL of cells at an OD600 of 0.3 to OD of 0.1 = [ (5mL*0.3)/0.1 - 5]  =10 mL
 +
</p>
 +
<li>Add cells to 96-well plate</li>
 +
<p>Pipette 100 uL of liquid cultures ot OD600= 0.1 into the corresponding wells,following 96-well microplate layout and culture tubes label.</p>
 +
<li>Set up the script in the plate reader and start experiment</li>
 +
<p>For Pyocyanin experiments:</p>
 +
<p>Set up a protocol that takes OD600 absorbance measurements and fluorescent intensity measurements every 10 minutes for 24 hours (288 cycles for 600 seconds cycles time). </p>
 +
<p>For ferro/ferricyanide experiments:
 +
</p>
 +
<p>
 +
For 200 uL of solution, we used a double orbital shaking rate of 200 rpm. Set temperature at 37°C.</p>
 +
<li>Data analysis</li>
 +
<p>Calculate mean and standard error for each set of replicates</p>
 +
 +
</ol>
 +
                </div>
 +
                <div class="clr"></div>
 
   <p2>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p2>
 
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</div>
 
</div>
Line 78: Line 689:
 
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Revision as of 21:39, 16 October 2018

Modelling



What is Modelling?

Constructing and manipulating abstract (mathematical and/or graphical) representations of economic, engineering, manufacturing, social, and other types of situations and natural phenomenon, simulated with the help of a computer system. Also called computer simulation.

Why is Modelling important in biological studies?

Computer modeling simulates the interaction among many different variables to provide scientists with a general idea of how a biological system may work.

Our Computer Models

Methods



Contents

Experiments

Assembling the Pixcell Constructs

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Description

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Materials

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Notes

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Procedure

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Characterizing Pixcell Constructs

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Description

This protocol describes how to measure E. coli growth and GFP expression over time with a plate reader, using the optical density at 600 nm as signal for cell density and excitation and emission wavelengths of 475. All characterization was performed using the FLUOstar BMG Labtech in greiner bioone F-bottom dark plates with clear bottom, to decrease fluorescence background. Each cycle of this experiment takes two days and has to be repeated two days, in order to have 2 biological and 2 technical replicates, for a total of 4 days and 4 96-well plates used.

Materials
  • Autoclaved LB media
  • Autoclaved 10x concentated LB media
  • 500 uM pyocyanin stock solution (from Sigma-Aldrich, CAS: No. 86-66-5)
  • 1250 mM potassium ferricyanide stock solution (from Sigma-Aldrich, CAS: No. 13746-66-2)
  • 500 mM ferrocyanide stock solution (from Sigma-Aldrich, CAS 14459-95-1 )
  • 2% Sodium Sulfite Solution
  • Agar plates with colonies of E. coli DJ901 strains with respective constructs: PixCell Patterning Circuit 1 (BBa_K2862021). PixCell Patterning Circuit 2 (BBa_K2862022), negative control (DJ901 WT) and positive control (constitutively expressed GFP).
  • 37°C shaking incubator
  • BMG Labtech FLUOstar plate reader
  • 4x Greiner BioOne 96-F 96-well microplate (black with clear bottom)
  • 14 mL culture tubes
  • autoclaved eppendorfs
Notes

Work in very sterile conditions. To prevent contaminations in the blanks, use triple antibiotics.
Store pyocyanin and ferrocyanide and ferricyanide solutions in the freezer until use.
We used 10x concentrated LB in order not to dilute nutrients at higher pyocyanin concentration; this was back diluted with autoclaved water. Mastemixes have double the working concentrations, as 100 uL of solution is then diluted with 100 uL of liquid cultures.

Procedure

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  1. Grow starter liquid cultures of cells (Day 0, 10-15 min preparation + overnight)

    2. Working in very sterile conditions, take 5 14mL culture tubes and prepare them according to table below:

    Label E. coli strain Construct Antibiotic (Ab) V Ab (uL) V LB (mL)
    PC1 DJ901 BBa_K2862021 Kan + Amp 5 + 5 5
    PC2 DJ901 BBa_K2862022 Kan + Amp 5 + 5 5
    - DJ901 none Kan 5 5
    + DJ901 GFP_Boo Kan + Chl 5 + 5 5
    Blanks None None Kan + Amp + Chl 5 + 5 5

    With a pipette tip pick a desired colony* from an agar plate and release the contaminated tip in the culture tip. Place inoculated culture tubes in a 37° C in a shaking incubator overnight (for faster growth the angle between the vertical axis of the tube and the shaking plane of the incubator should be of 45°)

  2. Prepare mastermixes (30 min)

    3. Mastermixes for each redox molecule concentration are prepared in 1.5 mL eppendorfs. The mastermixes are enough to fill 2 96-well microplates with 100 uL of solutions in each well, in order to have two replicate of the same experiment. Take 8 autoclaved 1.5 mL eppendorfs and prepare them as described in the table below.

    Mastermizes for Day 1 and Day 2 :

    Condition V Pyo (uL) Kan (uL) LB 10x uL Autoclaved Water (uL) Tot Vol (uL)
    A = 0 uM Pyo 0 2.44 122 1098 1220
    B = 0.01 uM Pyo 0.0976 2.44 122 1097.9024 1220
    C = 0.025 uM Pyo 0.244 2.44 122 1097.756 1220
    D = 0.05 uM Pyo 0.488 2.44 122 1097.512 1220
    E = 0.1 uM Pyo 0.976 2.44 122 1097.024 1220
    F = 0.25 uM Pyo 2.44 2.44 122 1095.56 1220
    G = 0.5 uM Pyo 4.88 2.44 122 1093.12 1220
    H = 1 uM Pyo 9.76 2.44 122 1088.24 1220

    Mastermixes for Day 3 and Day 4:

    Condition V Pyo (uL) Kan (uL) LB 10x uL Autoclaved Water (uL) Tot Vol (uL)
    A = 2.5 uM Pyo 24.4 2.44 122 1073.6 1220
    B = 5 uM Pyo 48.8 2.44 122 1049.2 1220
    C = 10 uM Pyo 97.6 2.44 122 1000.4 1220
    D = 25 uM Pyo 244 2.44 122 854 1220
    E = 50 uM Pyo 488 2.44 122 610 1220
    F = 100 uM Pyo 976 2.44 122 122 1220
  3. Fill 96-well plate with media solutions

    4. Add 100 uL of media in the desired wells, according following the label on mastermixes and table below:

    1 2 3 4 5 6 7 8 9 10 11 12
    A PC1 PC1 BA PC2 PC2 BA - - BA + + BA
    B PC1 PC1 BB PC2 PC2 BB - - BB + + BB
    C PC1 PC1 BC PC2 PC2 BC - - BC + + BC
    D PC1 PC1 BD PC2 PC2 BD - - BD + + BD
    E PC1 PC1 BE PC2 PC2 BE - - BE + + BE
    F PC1 PC1 BF PC2 PC2 BF - - BF + + BF
    G PC1 PC1 BG PC2 PC2 BG - - BG + + BG
    H PC1 PC1 BH PC2 PC2 BH - - BH + + BH
  4. OD600 matching

    5. Once the cultures tubes in the shaking incubator appear cloudy, take them out and in a fresh microplate add 100 uL of each sample into a well. Add 100 uL of LB + antibiotic into a blank well. Take endpoint measurement of the filled wells and blank them with LB+antibiotic. Note down the OD600 value of the wells and dilute them to the desired initial OD600 (a value of 0.1 is suggested for a volume of 100 uL

    Cell dilution calculator: Volume of LB+Antibiotic to add in culture tube = [( Vcells * ODcell)/desired OD] - Vcells E.g. to dilute 5mL of cells at an OD600 of 0.3 to OD of 0.1 = [ (5mL*0.3)/0.1 - 5] =10 mL

  5. Add cells to 96-well plate

    6. Pipette 100 uL of liquid cultures ot OD600= 0.1 into the corresponding wells,following 96-well microplate layout and culture tubes label.

  6. Set up the script in the plate reader and start experiment

    7. For Pyocyanin experiments:

    Set up a protocol that takes OD600 absorbance measurements and fluorescent intensity measurements every 10 minutes for 24 hours (288 cycles for 600 seconds cycles time).

    For ferro/ferricyanide experiments:

    For 200 uL of solution, we used a double orbital shaking rate of 200 rpm. Set temperature at 37°C.

  7. Data analysis

    8. Calculate mean and standard error for each set of replicates

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1 state patterning

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2 state patterning

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3 state patterning

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