http://2009.igem.org/wiki/index.php?title=Team:NTU-Singapore/Project/Prototype/Degrade&feed=atom&action=historyTeam:NTU-Singapore/Project/Prototype/Degrade - Revision history2024-03-29T10:25:30ZRevision history for this page on the wikiMediaWiki 1.16.5http://2009.igem.org/wiki/index.php?title=Team:NTU-Singapore/Project/Prototype/Degrade&diff=168497&oldid=prevZhumeng0331: /* SDS PAGE */2009-10-22T03:41:40Z<p><span class="autocomment">SDS PAGE</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>'''Fig 1''' - ''<del class="diffchange diffchange-inline">Positive controls of </del>SDS-PAGE <del class="diffchange diffchange-inline">show that SDS</del>-<del class="diffchange diffchange-inline">PAGE is applicable to identify proteins expressed in E. coli without purification</del>''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''Fig 1''' - ''SDS-PAGE <ins class="diffchange diffchange-inline">shows successful expression of positive controls of GFP (26.8 kDa) and β</ins>-<ins class="diffchange diffchange-inline">galacosidase (121 kDa)</ins>''</div></td></tr>
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</table>Zhumeng0331http://2009.igem.org/wiki/index.php?title=Team:NTU-Singapore/Project/Prototype/Degrade&diff=168360&oldid=prevZhumeng0331: /* SDS PAGE */2009-10-22T03:38:28Z<p><span class="autocomment">SDS PAGE</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">We saw inconclusive bands from SDS-PAGE. The reason may due to low expression level of CHE.</del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Cholesterol Assay Kit ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Cholesterol Assay Kit ====</div></td></tr>
</table>Zhumeng0331http://2009.igem.org/wiki/index.php?title=Team:NTU-Singapore/Project/Prototype/Degrade&diff=168122&oldid=prevIandravid at 03:32, 22 October 20092009-10-22T03:32:49Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== pLacI Characterization ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== pLacI Characterization ====</div></td></tr>
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</table>Iandravidhttp://2009.igem.org/wiki/index.php?title=Team:NTU-Singapore/Project/Prototype/Degrade&diff=167947&oldid=prevIandravid at 03:26, 22 October 20092009-10-22T03:26:23Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Test construct ('''pLac-RBS-CHE-T''') is constructed for the above mentioned experiments.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Test construct ('''pLac-RBS-CHE-T''') is constructed for the above mentioned experiments.</div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">==== pLacI Characterization ====</ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">'''Characterization of pLacI - GFP'''</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><span class="grey">Our degradation device is under the control inducible promoter, pLacI. As shown above, pLacI responses to increasing concentrations of IPTG within 1-3mM ranges. Hence a predictable working promoter gave us more confidence in carrying on with the expression of CHE. </span></ins></div></td></tr>
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</table>Iandravidhttp://2009.igem.org/wiki/index.php?title=Team:NTU-Singapore/Project/Prototype/Degrade&diff=167838&oldid=prevZhumeng0331: /* Cholesterol Assay Kit */2009-10-22T03:22:06Z<p><span class="autocomment">Cholesterol Assay Kit</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Fig. 6 Differences are observed between cells expressing CHE and cells not expressing CHE. Negative control denotes signals come from cells expressing CHE, but without the enzyme substrate. That is signals naturally generated by cell lysate as described in the former sections. Background is fluorescence emitted from samples without both enzyme substrate and cell lysate. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Fig. 6 Differences are observed between cells expressing CHE and cells not expressing <ins class="diffchange diffchange-inline">CHE. The insignificant differences may due to low expression level of </ins>CHE. Negative control denotes signals come from cells expressing CHE, but without the enzyme substrate. That is signals naturally generated by cell lysate as described in the former sections. Background is fluorescence emitted from samples without both enzyme substrate and cell lysate. </div></td></tr>
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</table>Zhumeng0331http://2009.igem.org/wiki/index.php?title=Team:NTU-Singapore/Project/Prototype/Degrade&diff=167446&oldid=prevZhumeng0331: /* Device Construction */2009-10-22T03:11:31Z<p><span class="autocomment">Device Construction</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We saw inconclusive bands from SDS-PAGE. The reason may due to low expression level of CHE.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We saw inconclusive bands from SDS-PAGE. The reason may due to low expression level of CHE.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Cholesterol Assay Kit ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Cholesterol Assay Kit ====</div></td></tr>
</table>Zhumeng0331http://2009.igem.org/wiki/index.php?title=Team:NTU-Singapore/Project/Prototype/Degrade&diff=167274&oldid=prevZhumeng0331: /* SDS PAGE */2009-10-22T03:06:33Z<p><span class="autocomment">SDS PAGE</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>SDS-PAGE is a routine technique employed to test the presence of certain proteins.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>SDS-PAGE is a routine technique employed to test the presence of certain proteins.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The expression of CHE in our '''Degradation device''' was confirmed by SDS-PAGE on a NuPAGE® Novex 4-12% Bis-Tris gel. Because no protein purification was carried out, a control sample that cannot produce CHE was run in parallel to verify its expression.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The expression of CHE in our '''Degradation device''' was confirmed by SDS-PAGE on a NuPAGE® Novex 4-12% Bis-Tris gel. Because no protein purification was carried out, a control sample that cannot produce CHE was run in parallel to verify its expression.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Although the above method is not as good as carrying out purified proteins with SDS-PAGE, positive controls have been done on cells expressing GFP (26.8 kDa) and β-galacosidase (121 kDa), and results show that this technique is applicable. The theoretical molecular weight of CHE, based on its protein sequence, is found to be 32.73 kDa. (Tools: [http://www.sciencegateway.org/tools/proteinmw.htm Protein Molecular Weight Calculator]). Protein ladder used is BenchMark™ Protein Ladder (Invitrogen Cat. No. 10747-012). </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Although the above method is not as good as carrying out purified proteins with SDS-PAGE, positive controls have been done on cells expressing GFP (26.8 kDa) and β-galacosidase (121 kDa), and results show that this technique is applicable. The theoretical molecular weight of CHE, based on its protein sequence, is found to be 32.73 kDa. (Tools: [http://www.sciencegateway.org/tools/proteinmw.htm Protein Molecular Weight Calculator]). Protein ladder used is BenchMark™ Protein Ladder (Invitrogen Cat. No. 10747-012). </div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">We saw inconclusive bands from SDS-PAGE. The reason may due to low expression level of CHE.</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Cholesterol Assay Kit ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Cholesterol Assay Kit ====</div></td></tr>
</table>Zhumeng0331http://2009.igem.org/wiki/index.php?title=Team:NTU-Singapore/Project/Prototype/Degrade&diff=166813&oldid=prevZhumeng0331: /* Cholesterol Assay Kit */2009-10-22T02:55:27Z<p><span class="autocomment">Cholesterol Assay Kit</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The Amplex® Red Cholesterol Assay Kit provides a simple fluorometric method for the sensitive quantitation of cholesterol using a fluorescence microplate reader or fluorometer. The assay is based on an enzyme-coupled reaction that detects free cholesterol concentration.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The Amplex® Red Cholesterol Assay Kit provides a simple fluorometric method for the sensitive quantitation of cholesterol using a fluorescence microplate reader or fluorometer. The assay is based on an enzyme-coupled reaction that detects free cholesterol concentration.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Cholesterol is oxidized by cholesterol oxidase to yield H<span class="sub">2</span>O<span class="sub">2</span> and the corresponding ketone product. The H<span class="sub">2</span>O<span class="sub">2</span> is then detected using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red reagent), a highly sensitive and stable probe for H<span class="sub">2</span>O<span class="sub">2</span>.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Cholesterol is oxidized by cholesterol oxidase to yield H<span class="sub">2</span>O<span class="sub">2</span> and the corresponding ketone product. The H<span class="sub">2</span>O<span class="sub">2</span> is then detected using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red reagent), a highly sensitive and stable probe for H<span class="sub">2</span>O<span class="sub">2</span>.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The theoretical enzyme activity, which needs the purification of CHE, is not carried out due to time limitations.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The theoretical enzyme activity, which needs the purification of CHE, is not carried out due to time limitations.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The first challenge we met in using the kit was to dissolve CEs, specifically cholesteryl linoleate, which is the substrate for our enzyme. CEs are insoluble in water, thus we tried to dissolve it in some popular organic solvent such as methanol, acetone, and DMES. However, it appeared that all those did not help.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The first challenge we met in using the kit was to dissolve CEs, specifically cholesteryl linoleate, which is the substrate for our enzyme. CEs are insoluble in water, thus we tried to dissolve it in some popular organic solvent such as methanol, acetone, and DMES. However, it appeared that all those did not help.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Interestingly, the negative controls that only contain isopropanol and assay reagents (both CHE+ and CHE-) also gave significant fluorescent signals. As H<span class="sub">2</span>O<span class="sub">2</span> cannot be easily generated in-vitro in this experiment, we therefore deduce that the reaction catalyzed by cholesterol oxidase is not specific, meaning that cholesterol oxidase can help oxidize a large range of substrate, especially those with hydroxyl group like alcohol. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Interestingly, the negative controls that only contain isopropanol and assay reagents (both CHE+ and CHE-) also gave significant fluorescent signals. As H<span class="sub">2</span>O<span class="sub">2</span> cannot be easily generated in-vitro in this experiment, we therefore deduce that the reaction catalyzed by cholesterol oxidase is not specific, meaning that cholesterol oxidase can help oxidize a large range of substrate, especially those with hydroxyl group like alcohol. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>If the above hypothesis is correct, we would expect interference signals generated from our controls, which are samples that do not express CHE, because there should be a lot of alcohols present in the soluble portion after sonication (most alcohols are soluble unlike CEs, unluckily).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>If the above hypothesis is correct, we would expect interference signals generated from our controls, which are samples that do not express CHE, because there should be a lot of alcohols present in the soluble portion after sonication (most alcohols are soluble unlike CEs, unluckily).</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Results show that both CHE+ and CHE- tests gave similar trend of signals versus time. And the steady state readings did not have significant difference. This confirms that cholesterol oxidase is a non-specific enzyme that oxidizes alcohols to ketones and H2O2, and there is not much CEs present in the soluble portion of cell lysate. (As expected, because CEs are very insoluble in water)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Results show that both CHE+ and CHE- tests gave similar trend of signals versus time. And the steady state readings did not have significant difference. This confirms that cholesterol oxidase is a non-specific enzyme that oxidizes alcohols to ketones and H2O2, and there is not much CEs present in the soluble portion of cell lysate. (As expected, because CEs are very insoluble in water)</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Although there is interference in our controls, the samples that contain CHE would also give the same signals as long as they are cultured and prepared identically. The interference signals from non-specific enzyme functionality can be generally assumed to be cancelled off.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Although there is interference in our controls, the samples that contain CHE would also give the same signals as long as they are cultured and prepared identically. The interference signals from non-specific enzyme functionality can be generally assumed to be cancelled off.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>However, in order to give significant differences between samples and controls which can be precisely used to show the functionality of CHE, high concentrations of CEs should be used as the enzymatic substrate. After a round of literature review and technical consultation, chloroform came to our eyes. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>However, in order to give significant differences between samples and controls which can be precisely used to show the functionality of CHE, high concentrations of CEs should be used as the enzymatic substrate. After a round of literature review and technical consultation, chloroform came to our eyes. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Chloroform is a well-known organic solvent, but is also very toxic. Cholestryl linoleate can dissolve in chloroform up to 100 mg/ml. As chloroform is immiscible with water and all our assay conditions are aqueous environment, chloroform dissolved with cholesteryl linoleate should be redissolved in an organic compound that is miscible with water (like acetone, NO alcohol!), or it should be homogenized with 0.9% NaCl solution by vigorous stirring.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Chloroform is a well-known organic solvent, but is also very toxic. Cholestryl linoleate can dissolve in chloroform up to 100 mg/ml. As chloroform is immiscible with water and all our assay conditions are aqueous environment, chloroform dissolved with cholesteryl linoleate should be redissolved in an organic compound that is miscible with water (like acetone, NO alcohol!), or it should be homogenized with 0.9% NaCl solution by vigorous stirring.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>With all the problems solved, we proceeded to test out whether our CHE construct works or not. Cells expressing CHE were cultured under the same conditions with control (cells with empty plasmids). Both cultures were harvested at the same OD, and lysed by sonication. The soluble portion of cell lysate was added to micro-plate reader, together with the substrate – cholesteryl linoleate.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>With all the problems solved, we proceeded to test out whether our CHE construct works or not. Cells expressing CHE were cultured under the same conditions with control (cells with empty plasmids). Both cultures were harvested at the same OD, and lysed by sonication. The soluble portion of cell lysate was added to micro-plate reader, together with the substrate – cholesteryl linoleate.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Results show that our CHE construct WORKS!'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Results show that our CHE construct WORKS!'''</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Fig. 6 Differences are observed between cells expressing CHE and cells not expressing CHE. Negative control denotes signals come from cells expressing CHE, but without the enzyme substrate. That is signals naturally generated by cell lysate as described in the former sections. Background is fluorescence emitted from samples without both enzyme substrate and cell lysate. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Fig. 6 Differences are observed between cells expressing CHE and cells not expressing CHE. Negative control denotes signals come from cells expressing CHE, but without the enzyme substrate. That is signals naturally generated by cell lysate as described in the former sections. Background is fluorescence emitted from samples without both enzyme substrate and cell lysate. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Literature / References ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Literature / References ==</div></td></tr>
</table>Zhumeng0331http://2009.igem.org/wiki/index.php?title=Team:NTU-Singapore/Project/Prototype/Degrade&diff=166731&oldid=prevZhumeng0331: /* Mechanism */2009-10-22T02:54:07Z<p><span class="autocomment">Mechanism</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The gene is designed to work under the control of promoter pTet. pTet is a well-known promoter that is constitutively ON and repressed by TetR. TetR repression is inhibited by the addition of tetracycline or its analog, aTc.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The gene is designed to work under the control of promoter pTet. pTet is a well-known promoter that is constitutively ON and repressed by TetR. TetR repression is inhibited by the addition of tetracycline or its analog, aTc.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since TetR production is controlled by pNO, and it is suppressed at low NO concentration (indicating inflammatory condition at the atherosclerosis site), the degrading device can be activated when NO concentration is low.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since TetR production is controlled by pNO, and it is suppressed at low NO concentration (indicating inflammatory condition at the atherosclerosis site), the degrading device can be activated when NO concentration is low.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>With the proper working of pNO and pTet inverter, CHE synthesis can accomplish designed objectives with both temporal and spatial specificity.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>With the proper working of pNO and pTet inverter, CHE synthesis can accomplish designed objectives with both temporal and spatial specificity.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== More about Cholesteryl Esterase ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== More about Cholesteryl Esterase ===</div></td></tr>
</table>Zhumeng0331http://2009.igem.org/wiki/index.php?title=Team:NTU-Singapore/Project/Prototype/Degrade&diff=166151&oldid=prevSamlotm: /* Modelling & Simulation */2009-10-22T02:42:43Z<p><span class="autocomment">Modelling & Simulation</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This is how increase in [NO] would affect CHE production. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This is how increase in [NO] would affect CHE production. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>''Deterministic'' simulation (left) & ''Stochastic'' simulation (right). A sharp decrease in production of CHE in response to increase in [NO]is predicted, followed by the attainment of steady state after 10 mins.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>''Deterministic'' simulation (left) & ''Stochastic'' simulation (right). A sharp decrease in production of CHE in response to increase in [NO] is predicted, followed by the attainment of steady state after 10 mins.</div></td></tr>
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</table>Samlotm