Team:Hong Kong CUHK/results

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<div id="middle">
<div id="containerX2" style="margin: 0px auto;">
<div id="containerX2" style="margin: 0px auto;">
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<div id="containerX4" style="margin: 0px auto;">
<div id="heading" style="margin: 0px auto;">
<div id="heading" style="margin: 0px auto;">
   <h2>Results</h2>
   <h2>Results</h2>
</div>
</div>
<div id="partable">
<div id="partable">
-
<strong></strong>
+
<h1>Characterization of Laccase</h1>
-
<h1> Laccase Activity Test</h1>
+
<h3>1. Biobrick DNA length verification</h3>
-
<p2><p>25-09-2013      </p>
+
<p align="center"><img src="https://static.igem.org/mediawiki/parts/f/fc/La_2.jpg" height="400"></p>
-
<p>Detection the conversion of the o-phenylenediamine (ELISA substrate) to colour product by laccase </p>
+
<p2><p align="center">Figure 1. Restriction digestion plasmid gel picture</p></p2>
-
<p>IPTG</p>
+
<h3>2. Laccase activity test by automatic kinetic spectrophotometric assay <br>
-
<p>Addition at OD=0.5</p>
+
using o-phenylenediamine</h3>
-
<p>12h incubation</p>
+
<p2>
-
<table cellspacing="0" cellpadding="0" border="1">
+
<p>Laccase is capable of oxidizing o-phenylenediamine (1,2-diaminobenzene) during the stopped-flow period. During oxidation, 2,3-diaminophenazin which can be determined spectrophotometrically, will be produced. Therefore, the kinetic spectrophotometric method was used here to detect laccase activity (Huang <em>et al</em>., 1996).</p>
-
  <col width="111" />
+
<p>After 10 to 12 hours of incubation at 37 °C, laccase showed maximum activity when 0.3 mM IPTG was used for introducing protein expression (Figure 2).</p>
-
  <col width="72" span="7" />
+
<p align="center"><a href="http://parts.igem.org/File:La1.jpg"><img alt="La1.jpg" src="https://static.igem.org/mediawiki/2013/a/a5/800px-La1.jpg" width="600" ></a></p>
-
  <tr>
+
<p align="center">Figure 2. Laccase activity monitoring</p>
-
    <td width="111">Laccase    biobrick</td>
+
-
    <td width="72">Absent</td>
+
-
    <td width="72">Present</td>
+
-
    <td width="72">Present</td>
+
-
    <td width="72">Present</td>
+
-
    <td width="72">Present</td>
+
-
    <td width="72">Present</td>
+
-
    <td width="72">Present</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>IPTG (mM)</td>
+
-
    <td align="right">0</td>
+
-
    <td align="right">0</td>
+
-
    <td align="right">0.01</td>
+
-
    <td align="right">0.03</td>
+
-
    <td align="right">0.1</td>
+
-
    <td align="right">0.3</td>
+
-
    <td align="right">1</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>OD492nm</td>
+
-
    <td align="right">0.0662</td>
+
-
    <td align="right">0.0691</td>
+
-
    <td align="right">0.0722</td>
+
-
    <td align="right">0.0735</td>
+
-
    <td align="right">0.0771</td>
+
-
    <td align="right">0.0999</td>
+
-
    <td align="right">0.0968</td>
+
-
  </tr>
+
-
</table>
+
-
<p><img src="https://static.igem.org/mediawiki/2013/9/9a/Curv1.png" alt="g1" width="367" height="281" /></p>
+
<p>&nbsp;</p>
<p>&nbsp;</p>
 +
<h3>3. Laccase function verification</h3>
 +
<p>
 +
Empty vector-bearing (EV) and laccase-overexpressing cells (T7-RBS-Laccase) were induced by 0.1 mM IPTG at OD600 = 0.4, followed by 100 mg/L BaP treatment for 24 h. Cells were removed and extracellular BaP was determined by OD389. Values represent the average of triplicate samples where error bars denote S.E.M., and normalized by the OD389 of cells treated with DMSO only. Paired <em>t</em>-test showed a significant difference between EV and laccase-overexpressing cells (<em>p</em> ≤ 0.01) (Figure 3).
 +
</p><p align="center"><a href="http://parts.igem.org/File:F4.jpg"><img alt="F4.jpg" src="https://static.igem.org/mediawiki/2013/8/85/El.JPG" width="348" height="240"></a></p>
 +
<p align="center">Figure 3. <em>E. coli</em> BL21 strain overexpressing laccase that degrades BaP</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
-
<table cellspacing="0" cellpadding="0" border="1">
+
<h1>Characterization of Catechol 1,2-dioxygenase</h1>
-
  <col width="72" />
+
<h3>1. Biobrick DNA length verification</h3>
-
  <col width="133" />
+
<p align="center"><img src="https://static.igem.org/mediawiki/parts/archive/8/8d/20130928021743%21Di.jpg" height="400"></p>
-
  <col width="111" />
+
<p align="center">Figure 4. Restriction digestion plasmid gel picture</p>
-
  <col width="72" span="11" />
+
<h3>2. Catechol 1,2-dioxygenase function verification by time series degradation assay</h3>
-
  <tr>
+
<p>Catechol 1,2-dioxygenase is resposible for phenol degredation (Naiem <em>et al</em>., 2011). Before it is integrated into our PAHs degrdation system, hydroquinone, also known as benzene-1,4-diol or quinol, which is an aromatic organic compound belonging to the phenol family, was used as a substrate to test Catechol 1,2-dioxygenase expression and activity.</p>
-
    <td width="109"></td>
+
<p>
-
    <td width="128">Wavelength</t>
+
   Time series analysis were performed. Empty vector-bearing (EV) and dioxygenase-overexpressing cells (T7-RBS-Dioxygenase) were induced by 0.3 mM IPTG at OD600 = 0.4, followed by 100 mg/L hydroquinone treatment. Cells were removed and extracellular hydroquinone was determined by OD298 at several intervals. Values represent the average of triplicate samples; error bars denote S.E.M. Paired <em>t</em>-test showed a significant difference in hydroquinone degradation trends between EV and dioxygenase-overexpressing cells (<em>p</em> ≤ 0.05) (Figure 2).</p>
-
    <td align="right" width="71">400</td>
+
<p align="center">
-
    <td align="right" width="73">405</td>
+
   <a href="http://parts.igem.org/File:Di_f2.jpg"><img alt="Di f2.jpg" src="https://static.igem.org/mediawiki/2013/1/16/Tsa.JPG" width="346"></a></p>
-
    <td align="right" width="72">410</td>
+
<p align="center">Figure 5. Time series analysis</p>
-
    <td align="right" width="72">415</td>
+
-
    <td align="right" width="72">420</td>
+
-
    <td align="right" width="72">425</td>
+
-
    <td align="right" width="72">430</td>
+
-
    <td align="right" width="72">435</td>
+
-
    <td align="right" width="72">440</td>
+
-
    <td align="right" width="72">445</td>
+
-
    <td align="right" width="73">450</td>
+
-
    <td align="right" width="74">455</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Laccase biobrick</td>
+
-
    <td>IPTG (mM)</td>
+
-
    <td>400nm</td>
+
-
    <td>405nm</td>
+
-
    <td>410nm</td>
+
-
    <td>415nm</td>
+
-
    <td>420nm</td>
+
-
    <td>425nm</td>
+
-
    <td>430nm</td>
+
-
    <td>435nm</td>
+
-
    <td>440nm</td>
+
-
    <td>445nm</td>
+
-
    <td>450nm</td>
+
-
    <td>455nm</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Absent</td>
+
-
    <td align="right">0</td>
+
-
    <td align="right">0.0778</td>
+
-
    <td align="right">0.076</td>
+
-
    <td align="right">0.0744</td>
+
-
    <td align="right">0.0736</td>
+
-
    <td align="right">0.0719</td>
+
-
    <td align="right">0.0713</td>
+
-
    <td align="right">0.0703</td>
+
-
    <td align="right">0.0694</td>
+
-
    <td align="right">0.0686</td>
+
-
    <td align="right">0.0686</td>
+
-
    <td align="right">0.068</td>
+
-
    <td align="right">0.0675</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0</td>
+
-
    <td align="right">0.0767</td>
+
-
    <td align="right">0.0751</td>
+
-
    <td align="right">0.0737</td>
+
-
    <td align="right">0.0725</td>
+
-
    <td align="right">0.0712</td>
+
-
    <td align="right">0.0706</td>
+
-
    <td align="right">0.0699</td>
+
-
    <td align="right">0.0692</td>
+
-
    <td align="right">0.0682</td>
+
-
    <td align="right">0.0687</td>
+
-
    <td align="right">0.0681</td>
+
-
    <td align="right">0.0683</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.01</td>
+
-
    <td align="right">0.0791</td>
+
-
    <td align="right">0.0773</td>
+
-
    <td align="right">0.0761</td>
+
-
    <td align="right">0.0751</td>
+
-
    <td align="right">0.0742</td>
+
-
    <td align="right">0.0731</td>
+
-
    <td align="right">0.0728</td>
+
-
    <td align="right">0.0721</td>
+
-
    <td align="right">0.071</td>
+
-
    <td align="right">0.0716</td>
+
-
    <td align="right">0.071</td>
+
-
    <td align="right">0.0714</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.03</td>
+
-
    <td align="right">0.0743</td>
+
-
    <td align="right">0.0728</td>
+
-
    <td align="right">0.0711</td>
+
-
    <td align="right">0.07</td>
+
-
    <td align="right">0.0693</td>
+
-
    <td align="right">0.0688</td>
+
-
    <td align="right">0.0681</td>
+
-
    <td align="right">0.0677</td>
+
-
    <td align="right">0.0675</td>
+
-
    <td align="right">0.068</td>
+
-
    <td align="right">0.0681</td>
+
-
    <td align="right">0.0692</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.1</td>
+
-
    <td align="right">0.0761</td>
+
-
    <td align="right">0.0743</td>
+
-
    <td align="right">0.0727</td>
+
-
    <td align="right">0.072</td>
+
-
    <td align="right">0.0703</td>
+
-
    <td align="right">0.0699</td>
+
-
    <td align="right">0.0689</td>
+
-
    <td align="right">0.0685</td>
+
-
    <td align="right">0.0684</td>
+
-
    <td align="right">0.069</td>
+
-
    <td align="right">0.07</td>
+
-
    <td align="right">0.0711</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.3</td>
+
-
    <td align="right">0.0964</td>
+
-
    <td align="right">0.0946</td>
+
-
    <td align="right">0.0931</td>
+
-
    <td align="right">0.0922</td>
+
-
    <td align="right">0.0909</td>
+
-
    <td align="right">0.0903</td>
+
-
    <td align="right">0.09</td>
+
-
    <td align="right">0.0901</td>
+
-
    <td align="right">0.0902</td>
+
-
    <td align="right">0.0917</td>
+
-
    <td align="right">0.0926</td>
+
-
    <td align="right">0.0943</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">1</td>
+
-
    <td align="right">0.0947</td>
+
-
    <td align="right">0.0927</td>
+
-
    <td align="right">0.0912</td>
+
-
    <td align="right">0.0901</td>
+
-
    <td align="right">0.0887</td>
+
-
    <td align="right">0.0881</td>
+
-
    <td align="right">0.0879</td>
+
-
    <td align="right">0.0873</td>
+
-
    <td align="right">0.0878</td>
+
-
    <td align="right">0.0891</td>
+
-
    <td align="right">0.0898</td>
+
-
    <td align="right">0.0916</td>
+
-
  </tr>
+
-
</table>
+
-
<table cellspacing="0" cellpadding="0" border="1">
+
-
  <col width="72" span="16" />
+
-
  <tr>
+
-
    <td width="109"></td>
+
-
    <td width="128">Wavelength</td>
+
-
    <td align="right" width="72">460</td>
+
-
    <td align="right" width="72">465</td>
+
-
    <td align="right" width="72">470</td>
+
-
    <td align="right" width="72">475</td>
+
-
    <td align="right" width="72">480</td>
+
-
    <td align="right" width="72">485</td>
+
-
    <td align="right" width="72">490</td>
+
-
    <td align="right" width="72">495</td>
+
-
    <td align="right" width="72">500</td>
+
-
    <td align="right" width="72">505</td>
+
-
    <td align="right" width="72">510</td>
+
-
    <td align="right" width="75">515</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Laccase biobrick</td>
+
-
    <td>IPTG (mM)</td>
+
-
    <td>460nm</td>
+
-
    <td>465nm</td>
+
-
    <td>470nm</td>
+
-
    <td>475nm</td>
+
-
    <td>480nm</td>
+
-
    <td>485nm</td>
+
-
    <td>490nm</td>
+
-
    <td>495nm</td>
+
-
    <td>500nm</td>
+
-
    <td>505nm</td>
+
-
    <td>510nm</td>
+
-
    <td>515nm</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Absent</td>
+
-
    <td align="right">0</td>
+
-
    <td align="right">0.0673</td>
+
-
    <td align="right">0.067</td>
+
-
    <td align="right">0.0667</td>
+
-
    <td align="right">0.0667</td>
+
-
    <td align="right">0.0667</td>
+
-
    <td align="right">0.0657</td>
+
-
    <td align="right">0.0652</td>
+
-
    <td align="right">0.0656</td>
+
-
    <td align="right">0.0646</td>
+
-
    <td align="right">0.0637</td>
+
-
    <td align="right">0.0629</td>
+
-
    <td align="right">0.0623</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0</td>
+
-
    <td align="right">0.0681</td>
+
-
    <td align="right">0.0677</td>
+
-
    <td align="right">0.0677</td>
+
-
    <td align="right">0.0675</td>
+
-
    <td align="right">0.0675</td>
+
-
    <td align="right">0.0674</td>
+
-
    <td align="right">0.068</td>
+
-
    <td align="right">0.0684</td>
+
-
    <td align="right">0.0672</td>
+
-
    <td align="right">0.0658</td>
+
-
    <td align="right">0.0635</td>
+
-
    <td align="right">0.0625</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.01</td>
+
-
    <td align="right">0.0712</td>
+
-
    <td align="right">0.0711</td>
+
-
    <td align="right">0.0707</td>
+
-
    <td align="right">0.0709</td>
+
-
    <td align="right">0.0716</td>
+
-
    <td align="right">0.0709</td>
+
-
    <td align="right">0.0711</td>
+
-
    <td align="right">0.0715</td>
+
-
    <td align="right">0.0699</td>
+
-
    <td align="right">0.068</td>
+
-
    <td align="right">0.0658</td>
+
-
    <td align="right">0.065</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.03</td>
+
-
    <td align="right">0.0696</td>
+
-
    <td align="right">0.0698</td>
+
-
    <td align="right">0.0703</td>
+
-
    <td align="right">0.0705</td>
+
-
    <td align="right">0.0712</td>
+
-
    <td align="right">0.0721</td>
+
-
    <td align="right">0.0726</td>
+
-
    <td align="right">0.0735</td>
+
-
    <td align="right">0.0701</td>
+
-
    <td align="right">0.0665</td>
+
-
    <td align="right">0.0621</td>
+
-
    <td align="right">0.0602</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.1</td>
+
-
    <td align="right">0.072</td>
+
-
    <td align="right">0.0724</td>
+
-
    <td align="right">0.0731</td>
+
-
    <td align="right">0.0735</td>
+
-
    <td align="right">0.0749</td>
+
-
    <td align="right">0.0758</td>
+
-
    <td align="right">0.0767</td>
+
-
    <td align="right">0.0773</td>
+
-
    <td align="right">0.0732</td>
+
-
    <td align="right">0.0686</td>
+
-
    <td align="right">0.0631</td>
+
-
    <td align="right">0.0606</td>
+
-
    </tr>
+
-
   <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.3</td>
+
-
    <td align="right">0.0962</td>
+
-
    <td align="right">0.0971</td>
+
-
    <td align="right">0.0974</td>
+
-
    <td align="right">0.0983</td>
+
-
    <td align="right">0.1002</td>
+
-
    <td align="right">0.1029</td>
+
-
    <td align="right">0.1046</td>
+
-
    <td align="right">0.1054</td>
+
-
    <td align="right">0.0987</td>
+
-
    <td align="right">0.0922</td>
+
-
    <td align="right">0.0838</td>
+
-
    <td align="right">0.08</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">1</td>
+
-
    <td align="right">0.0932</td>
+
-
    <td align="right">0.0937</td>
+
-
    <td align="right">0.0943</td>
+
-
    <td align="right">0.0951</td>
+
-
    <td align="right">0.0973</td>
+
-
    <td align="right">0.099</td>
+
-
    <td align="right">0.1004</td>
+
-
    <td align="right">0.101</td>
+
-
    <td align="right">0.0954</td>
+
-
    <td align="right">0.0893</td>
+
-
    <td align="right">0.0815</td>
+
-
    <td align="right">0.0774</td>
+
-
    </tr>
+
-
</table>
+
-
<table cellpadding="0" cellspacing="0" border="1">
+
-
  <col width="72" span="16" />
+
-
  <tr>
+
-
    <td width="109"></td>
+
-
    <td width="128">Wavelength</td>
+
-
    <td align="right" width="72">520</td>
+
-
    <td align="right" width="71">525</td>
+
-
    <td align="right" width="73">530</td>
+
-
    <td align="right" width="73">535</td>
+
-
    <td align="right" width="71">540</td>
+
-
    <td align="right" width="72">545</td>
+
-
    <td align="right" width="71">550</td>
+
-
    <td align="right" width="74">555</td>
+
-
    <td align="right" width="72">560</td>
+
-
    <td align="right" width="71">565</td>
+
-
    <td align="right" width="72">570</td>
+
-
    <td align="right" width="74">575</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Laccase biobrick</td>
+
-
    <td>IPTG (mM)</td>
+
-
    <td>520nm</td>
+
-
    <td>525nm</td>
+
-
    <td>530nm</td>
+
-
    <td>535nm</td>
+
-
    <td>540nm</td>
+
-
    <td>545nm</td>
+
-
    <td>550nm</td>
+
-
    <td>555nm</td>
+
-
    <td>560nm</td>
+
-
    <td>565nm</td>
+
-
    <td>570nm</td>
+
-
    <td>575nm</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Absent</td>
+
-
    <td align="right">0</td>
+
-
    <td align="right">0.0615</td>
+
-
    <td align="right">0.0617</td>
+
-
    <td align="right">0.0616</td>
+
-
    <td align="right">0.0613</td>
+
-
    <td align="right">0.0607</td>
+
-
    <td align="right">0.0606</td>
+
-
    <td align="right">0.0607</td>
+
-
    <td align="right">0.0606</td>
+
-
    <td align="right">0.0602</td>
+
-
    <td align="right">0.0599</td>
+
-
    <td align="right">0.0596</td>
+
-
    <td align="right">0.0591</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0</td>
+
-
    <td align="right">0.0617</td>
+
-
    <td align="right">0.0611</td>
+
-
    <td align="right">0.061</td>
+
-
    <td align="right">0.0605</td>
+
-
    <td align="right">0.0595</td>
+
-
    <td align="right">0.0596</td>
+
-
    <td align="right">0.0593</td>
+
-
    <td align="right">0.0593</td>
+
-
    <td align="right">0.0591</td>
+
-
    <td align="right">0.0582</td>
+
-
    <td align="right">0.0579</td>
+
-
    <td align="right">0.0575</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.01</td>
+
-
    <td align="right">0.0641</td>
+
-
    <td align="right">0.064</td>
+
-
    <td align="right">0.0636</td>
+
-
    <td align="right">0.0628</td>
+
-
    <td align="right">0.0622</td>
+
-
    <td align="right">0.0621</td>
+
-
    <td align="right">0.0615</td>
+
-
    <td align="right">0.0616</td>
+
-
    <td align="right">0.0614</td>
+
-
    <td align="right">0.0608</td>
+
-
    <td align="right">0.0607</td>
+
-
    <td align="right">0.06</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.03</td>
+
-
    <td align="right">0.0586</td>
+
-
    <td align="right">0.0579</td>
+
-
    <td align="right">0.0573</td>
+
-
    <td align="right">0.0564</td>
+
-
    <td align="right">0.0559</td>
+
-
    <td align="right">0.0555</td>
+
-
    <td align="right">0.055</td>
+
-
    <td align="right">0.0552</td>
+
-
    <td align="right">0.0547</td>
+
-
    <td align="right">0.0543</td>
+
-
    <td align="right">0.0539</td>
+
-
    <td align="right">0.0537</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.1</td>
+
-
    <td align="right">0.0585</td>
+
-
    <td align="right">0.0577</td>
+
-
    <td align="right">0.0572</td>
+
-
    <td align="right">0.0562</td>
+
-
    <td align="right">0.0553</td>
+
-
    <td align="right">0.055</td>
+
-
    <td align="right">0.0546</td>
+
-
    <td align="right">0.0541</td>
+
-
    <td align="right">0.0535</td>
+
-
    <td align="right">0.0531</td>
+
-
    <td align="right">0.0527</td>
+
-
    <td align="right">0.0525</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.3</td>
+
-
    <td align="right">0.077</td>
+
-
    <td align="right">0.076</td>
+
-
    <td align="right">0.0744</td>
+
-
    <td align="right">0.0729</td>
+
-
    <td align="right">0.0723</td>
+
-
    <td align="right">0.0713</td>
+
-
    <td align="right">0.0707</td>
+
-
    <td align="right">0.0703</td>
+
-
    <td align="right">0.07</td>
+
-
    <td align="right">0.0694</td>
+
-
    <td align="right">0.0686</td>
+
-
    <td align="right">0.0685</td>
+
-
    </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">1</td>
+
-
    <td align="right">0.0749</td>
+
-
    <td align="right">0.0738</td>
+
-
    <td align="right">0.073</td>
+
-
    <td align="right">0.0712</td>
+
-
    <td align="right">0.07</td>
+
-
    <td align="right">0.0698</td>
+
-
    <td align="right">0.0693</td>
+
-
    <td align="right">0.069</td>
+
-
    <td align="right">0.0687</td>
+
-
    <td align="right">0.0682</td>
+
-
    <td align="right">0.0681</td>
+
-
    <td align="right">0.0678</td>
+
-
    </tr>
+
-
</table>
+
-
<table cellspacing="0" cellpadding="0" border="1">
+
-
  <col width="72" span="13" />
+
-
  <tr>
+
-
    <td width="109"></td>
+
-
    <td width="128">Wavelength</td>
+
-
    <td align="right" width="72">580</td>
+
-
    <td align="right" width="72">585</td>
+
-
    <td align="right" width="72">590</td>
+
-
    <td align="right" width="72">595</td>
+
-
    <td align="right" width="72">600</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Laccase biobrick</td>
+
-
    <td>IPTG (mM)</td>
+
-
    <td>580nm</td>
+
-
    <td>585nm</td>
+
-
    <td>590nm</td>
+
-
    <td>595nm</td>
+
-
    <td>600nm</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Absent</td>
+
-
    <td align="right">0</td>
+
-
    <td align="right">0.0584</td>
+
-
    <td align="right">0.0585</td>
+
-
    <td align="right">0.0582</td>
+
-
    <td align="right">0.0577</td>
+
-
    <td align="right">0.0579</td>
+
-
   </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0</td>
+
-
    <td align="right">0.0573</td>
+
-
    <td align="right">0.0571</td>
+
-
    <td align="right">0.057</td>
+
-
    <td align="right">0.0564</td>
+
-
    <td align="right">0.0565</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.01</td>
+
-
    <td align="right">0.0595</td>
+
-
    <td align="right">0.0594</td>
+
-
    <td align="right">0.0595</td>
+
-
    <td align="right">0.0589</td>
+
-
    <td align="right">0.0591</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.03</td>
+
-
    <td align="right">0.0533</td>
+
-
    <td align="right">0.0532</td>
+
-
    <td align="right">0.053</td>
+
-
    <td align="right">0.0522</td>
+
-
    <td align="right">0.0528</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.1</td>
+
-
    <td align="right">0.0524</td>
+
-
    <td align="right">0.052</td>
+
-
    <td align="right">0.0512</td>
+
-
    <td align="right">0.0512</td>
+
-
    <td align="right">0.0509</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">0.3</td>
+
-
    <td align="right">0.0678</td>
+
-
    <td align="right">0.0675</td>
+
-
    <td align="right">0.0671</td>
+
-
    <td align="right">0.0668</td>
+
-
    <td align="right">0.0663</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td>Present</td>
+
-
    <td align="right">1</td>
+
-
    <td align="right">0.067</td>
+
-
    <td align="right">0.0665</td>
+
-
    <td align="right">0.0665</td>
+
-
    <td align="right">0.066</td>
+
-
    <td align="right">0.0659</td>
+
-
  </tr>
+
-
</table>
+
-
<p><img src="https://static.igem.org/mediawiki/2013/3/3d/Curv2.png" alt="g2" width="726" height="289" /></p>
+
<p>&nbsp;</p>
<p>&nbsp;</p>
-
<p>&nbsp;</p>
 
-
<table cellspacing="0" cellpadding="0" border="1">
 
-
  <col width="72" span="5"/>
 
-
  <tr>
 
-
    <td width="113"></td>
 
-
    <td colspan="6">Calculate    the activity of laccase from acitivty of peroxidase</td>
 
-
  </tr>
 
-
  <tr>
 
-
    <td>&nbsp;</td>
 
-
    <td colspan="6">1U is defined as μmol    conversion of products per minute</td>
 
-
  </tr>
 
-
  <tr>
 
-
    <td align="right">Control</td>
 
-
    <td width="75" align="right">0</td>
 
-
    <td width="75" align="right">0.05</td>
 
-
    <td width="75" align="right">0.1</td>
 
-
    <td width="75" align="right">1</td>
 
-
    <td width="75" align="right">5</td>
 
-
 
 
-
  </tr>
 
-
  <tr>
 
-
    <td align="right">Peroxidase(mU)</td>
 
-
    <td align="right">0.0609</td>
 
-
    <td align="right">0.0744</td>
 
-
    <td align="right">0.0868</td>
 
-
    <td align="right">0.2405</td>
 
-
    <td align="right">0.4129</td>
 
-
   
 
-
    </tr>
 
-
</table>
 
-
<p><img src="https://static.igem.org/mediawiki/2013/1/1e/Curv3.png" alt="g3" width="435" height="334" /></p>
 
-
 
-
<h1>E.  coli BL21 transformed with catechol 1,2-dioxygenase</h1>
 
-
<p>wild type E. coli BL21</p>
 
-
<p> <br />
 
-
  0        50.00           50.00<br />
 
-
  1        16.54           50.00<br />
 
-
  2        0.00  37.69</p>
 
-
<p><img src="https://static.igem.org/mediawiki/2013/a/ab/Curv4.png" alt="graph" width="622" height="340" /></p>
 
-
<p>Pearson's Chi-squared test</p>
 
-
<p>data:   a<br />
 
-
  X-squared = 33.8347, df = 2, p-value =  4.497e-08</p>
 
<h1>Cell  Viability Experiment with Voltage </h1>
<h1>Cell  Viability Experiment with Voltage </h1>
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   <p>This experiment is designed to investigate  the BL21 viability when voltage is applied. This is necessary for the voltage  sensor experiments in our project. The protocol of the following experiment can  be found here.(hyperlink to <a href="http://www.facebook.com/l.php?u=http%3A%2F%2F2013.igem.org%2FTeam%3AHong_Kong_CUHK%2Fprotocol&amp;h=dAQGS0x-i">https://2013.igem.org/Team:Hong_Kong_CUHK/protocol</a>)</p>
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   <p>This experiment is designed to investigate  the BL21 viability when voltage is applied. This is necessary for the voltage  sensor experiments in our project. The protocol of the following experiment can  be found <a href="https://2013.igem.org/Team:Hong_Kong_CUHK/protocol">here</a>.</p>
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<p><img border="0" width="552" height="725" src="https://static.igem.org/mediawiki/igem.org/2/2b/G5.png" alt="Macintosh HD:Users:melisajunata:Desktop:Cell Viability Experiment with Voltage.png" /></p>
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  <p align="center">(a)<img width="300" height="225" src="https://static.igem.org/mediawiki/2013/a/a3/Plate.jpg" alt="Macintosh HD:Users:melisajunata:Desktop:picts result:IMG_2676.JPG" />
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<p>&nbsp;</p>
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  <img width="300" height="225" src="https://static.igem.org/mediawiki/2013/0/0b/CVResult2.jpg" alt="Macintosh HD:Users:melisajunata:Desktop:picts result:IMG_2677.JPG" />(b)</p>
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<p>&nbsp;</p>
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  <p align="center">(c)<img width="300" height="225" src="https://static.igem.org/mediawiki/2013/0/01/CVResult3.jpg" alt="Macintosh HD:Users:melisajunata:Desktop:picts result:IMG_2678.JPG" />
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<p><img border="0" width="553" height="264" src="https://static.igem.org/mediawiki/2013/5/5e/Curv6.jpg" alt="Macintosh HD:Users:melisajunata:Desktop:Untitled.jpg" /></p>
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  <img width="300" height="225" src="https://static.igem.org/mediawiki/2013/a/aa/CVResult4.jpg" alt="Macintosh HD:Users:melisajunata:Desktop:picts result:IMG_2679.JPG" />(d)</p>
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<p>The difference in optical density (OD) before and after experiment determines the number of cells that grow during the  experimental period.</p>
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  <p align="center">(e)<img width="300" height="225" src="https://static.igem.org/mediawiki/2013/f/f0/CVResult5.jpg" alt="Macintosh HD:Users:melisajunata:Desktop:picts result:IMG_2680.JPG" /></p>
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<p>The results show that the control group,  BL21 E.coli without pSB1C3+RFP transformation survive better when voltage is  applied as they are shaken in the incubator (25°C). On the other hand, the  experimental group, BL212 E.coli with pSB1C3 + RFP shows a lower survival rate  when voltage is applied and shaken inside the incubator (25°C).</p>
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  <p align="center">Figure 6. Cell growth after treatment (a) Spread plate result with 0V applied. (b) Spread plate result with 9V applied. (c) Spread plate result with 18V applied. (d) Spread plate result with 27V applied. (e) Spread plate result with 45V applied.</p>
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<p>Despite of the voltage being positive or  negative, both groups show higher survival rate when lower magnitude voltage is  applied, as shown on the graph.</p>
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  <p>The result suggests that applying a continuous DC Voltage for 24 hours to the samples won't kill the cells. Colonies are growing in all plates. Decreasing quantity of colonies is  only due to the increasing dilution factor. </p>
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<p>&nbsp;</p>
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   Address: Rm. 184, Science Centre, CUHK <br/>
   Address: Rm. 184, Science Centre, CUHK <br/>
   Email: kingchan@cuhk.edu.hk  Tel: (852)-39434420  Fax: (852)-26037246
   Email: kingchan@cuhk.edu.hk  Tel: (852)-39434420  Fax: (852)-26037246
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Latest revision as of 03:52, 29 October 2013

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