Team:Hong Kong CUHK/results

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<h1>Characterization of Laccase</h1>
<h1>Characterization of Laccase</h1>
<h3>1. Biobrick DNA length verification</h3>
<h3>1. Biobrick DNA length verification</h3>
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<p align="center"><img src="https://static.igem.org/mediawiki/2013/d/dd/20051.png" height="300"></p>
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<p align="center"><img src="https://static.igem.org/mediawiki/parts/f/fc/La_2.jpg" height="400"></p>
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<p2><p align="center">Figure 1. Restriction digestion plasmid gel picture</p></p2>
<h3>2. Laccase activity test by automatic kinetic spectrophotometric assay <br>
<h3>2. Laccase activity test by automatic kinetic spectrophotometric assay <br>
using o-phenylenediamine</h3>
using o-phenylenediamine</h3>
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<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>
<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>
<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>
<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>
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<p align="center">Figure 1. Laccase Activity Monitoring</p>
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<p align="center">Figure 2. Laccase activity monitoring</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
<h3>3. Laccase function verification</h3>
<h3>3. Laccase function verification</h3>
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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).
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><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>
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<p align="center">Figure 2. <em>E. coli</em> BL21 strain overexpressing laccase that degrades BaP</p>
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<p align="center">Figure 3. <em>E. coli</em> BL21 strain overexpressing laccase that degrades BaP</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
<h1>Characterization of Catechol 1,2-dioxygenase</h1>
<h1>Characterization of Catechol 1,2-dioxygenase</h1>
<h3>1. Biobrick DNA length verification</h3>
<h3>1. Biobrick DNA length verification</h3>
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<p align="center"><img src="https://static.igem.org/mediawiki/2013/e/e6/20032.png" height="300"></p>
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<p align="center"><img src="https://static.igem.org/mediawiki/parts/archive/8/8d/20130928021743%21Di.jpg" height="400"></p>
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<p align="center">Figure 4. Restriction digestion plasmid gel picture</p>
<h3>2. Catechol 1,2-dioxygenase function verification by time series degradation assay</h3>
<h3>2. Catechol 1,2-dioxygenase function verification by time series degradation assay</h3>
<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>
<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>
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<p align="center">
<p align="center">
   <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>
   <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>
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<p align="center">Figure 3. Time series analysis</p>
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<p align="center">Figure 5. Time series analysis</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
<h1>Cell  Viability Experiment with Voltage </h1>
<h1>Cell  Viability Experiment with Voltage </h1>
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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>
   <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>
   <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>
   <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 align="center">Figure 5. (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 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>From these results, it can be said that by  applying a continuous DC Voltage for 24 hours to the samples do not kill the cell. Colonies are growing in all plates. Decreasing quantity of colonies is  only due to the increasing dilution factor. </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>
</p2>
</p2>
</div></div>
</div></div>

Latest revision as of 03:52, 29 October 2013

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