http://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Speed_Control_Methodology&feed=atom&action=historyTeam:HKU-HKBU/Speed Control Methodology - Revision history2024-03-29T12:30:28ZRevision history for this page on the wikiMediaWiki 1.16.5http://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Speed_Control_Methodology&diff=166801&oldid=prevYinanZhang at 02:55, 22 October 20092009-10-22T02:55:15Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>A <del class="diffchange diffchange-inline">psim6 </del>plasmid, which could help Recombineering process, was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of YBS01 by electroporation. After recovering with SOC solution for 30 minutes, they were spread to Agar plates with Ampicillin resistance. Then one single colony on the plate was picked up for [[Team:HKU-HKBU/Protocols#Pre-culture | pre-culture]].</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>A <ins class="diffchange diffchange-inline">pSim6 </ins>plasmid, which could help Recombineering process, was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of YBS01 by electroporation. After recovering with SOC solution for 30 minutes, they were spread to Agar plates with Ampicillin resistance. Then one single colony on the plate was picked up for [[Team:HKU-HKBU/Protocols#Pre-culture | pre-culture]].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 2===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 2===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>A DNA fragment with <del class="diffchange diffchange-inline">homologeous </del>arms made by PCR using specific primers was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the YBS01 with psim6. After recovering with SOC solution for 120 minutes, they were spread to Agar plates with chloramphenicol resistance. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>A DNA fragment with <ins class="diffchange diffchange-inline">homologous </ins>arms made by PCR using specific primers was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the YBS01 with psim6. After recovering with SOC solution for 120 minutes, they were spread to Agar plates with chloramphenicol resistance. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 3===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 3===</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 2===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 2===</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The <del class="diffchange diffchange-inline">bacteria </del>[[Team:HKU-HKBU/Protocols#Bacteria_Lysis | bacteria lysis]] underwent to release the protein samples. [[Team:HKU-HKBU/Protocols#BCA_Quantification_Analysis | BCA quantification analysis]] was done to detect the concentrations of these protein samples. After analyzing the concentrations of each protein samples, we used the formula: total amount of protein= sample concentration*sample volume to adjust the sample volume to the same loading volume by adding the mixture buffer. Equal loading of protein samples were ensured in [[Team:HKU-HKBU/Protocols#Western_Blotting | Western blotting]] after these adjustments.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The [[Team:HKU-HKBU/Protocols#Bacteria_Lysis | bacteria lysis]] underwent to release the protein samples. [[Team:HKU-HKBU/Protocols#BCA_Quantification_Analysis | BCA quantification analysis]] was done to detect the concentrations of these protein samples. After analyzing the concentrations of each protein samples, we used the formula: total amount of protein= sample concentration*sample volume to adjust the sample volume to the same loading volume by adding the mixture buffer. Equal loading of protein samples were ensured in [[Team:HKU-HKBU/Protocols#Western_Blotting | Western blotting]] after these adjustments.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 3===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 3===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A wider range of aTc concentrations were added to YBE02 and YBS02 with the plasmid [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 '''BBa_K283002'''] </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A wider range of aTc concentrations were added to YBE02 and YBS02 with the plasmid [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 '''BBa_K283002'''] </div></td></tr>
<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;"></del></div></td><td colspan="2"> </td></tr>
<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;"></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>{{Team:HKU-HKBU/footer}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:HKU-HKBU/footer}}</div></td></tr>
</table>YinanZhanghttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Speed_Control_Methodology&diff=148635&oldid=prevYubin: /* Step 3 */2009-10-21T16:53:19Z<p><span class="autocomment">Step 3</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A wider range of aTc concentrations were added to YBE02 and YBS02 with the plasmid [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 '''BBa_K283002'''] </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A wider range of aTc concentrations were added to YBE02 and YBS02 with the plasmid [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 '''BBa_K283002'''] </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">{| align="center" border="1px" cellpadding="4px"</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">! colspan="6" | IPTG concentration(uM)</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">|-</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">| 0 || 1 || 2 || 4 || 8 || 12</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">|}</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:HKU-HKBU/footer}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:HKU-HKBU/footer}}</div></td></tr>
</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Speed_Control_Methodology&diff=148629&oldid=prevYubin: /* Step 1 */2009-10-21T16:53:07Z<p><span class="autocomment">Step 1</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>YBE02 and YBS02 (delta ''cheZ'') strain were used to test whether the inducible regulation of ''cheZ'' would succeed. With no background expression of ''cheZ'', we would be able to achieve precise control over intracellular cheZ level through regulated gene expression. The plasmid [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283014 '''BBa_K283014'''] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 '''BBa_K283002'''] was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of and YBE02 and YBS02 of ''cheZ'' expression was induced through incubating with different concentrations of IPTG or aTc and various induced time. The samples were gathered at specific time and with different IPTG or aTc concentrations as follows.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>YBE02 and YBS02 (delta ''cheZ'') strain were used to test whether the inducible regulation of ''cheZ'' would succeed. With no background expression of ''cheZ'', we would be able to achieve precise control over intracellular cheZ level through regulated gene expression. The plasmid [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283014 '''BBa_K283014'''] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 '''BBa_K283002'''] was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of and YBE02 and YBS02 of ''cheZ'' expression was induced through incubating with different concentrations of IPTG or aTc and various induced time. The samples were gathered at specific time and with different IPTG or aTc concentrations as follows.</div></td></tr>
<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;"></del></div></td><td colspan="2"> </td></tr>
<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;">{| align="center" border="1px" cellpadding="4px"</del></div></td><td colspan="2"> </td></tr>
<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;">! rowspan="2" | Culture Time !! colspan="4" | IPTG or aTc concentration</del></div></td><td colspan="2"> </td></tr>
<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;">|-</del></div></td><td colspan="2"> </td></tr>
<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;">! 0uM !! 1uM !! 2uM !! 3.3uM</del></div></td><td colspan="2"> </td></tr>
<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;">|-</del></div></td><td colspan="2"> </td></tr>
<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;">| 1hr</del></div></td><td colspan="2"> </td></tr>
<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;">|-</del></div></td><td colspan="2"> </td></tr>
<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;">| 2hr</del></div></td><td colspan="2"> </td></tr>
<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;">|-</del></div></td><td colspan="2"> </td></tr>
<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;">| 3hr</del></div></td><td colspan="2"> </td></tr>
<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;">|-</del></div></td><td colspan="2"> </td></tr>
<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;">| 24hr</del></div></td><td colspan="2"> </td></tr>
<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;">|}</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>===Step 2===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 2===</div></td></tr>
</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Speed_Control_Methodology&diff=148620&oldid=prevYubin at 16:52, 21 October 20092009-10-21T16:52:35Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 3===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 3===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>A wider range of <del class="diffchange diffchange-inline">IPTG </del>concentrations were added to <del class="diffchange diffchange-inline">MG3 bacteria </del>with the plasmid <del class="diffchange diffchange-inline">plac-his-cheZ-cm</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>A wider range of <ins class="diffchange diffchange-inline">aTc </ins>concentrations were added to <ins class="diffchange diffchange-inline">YBE02 and YBS02 </ins>with the plasmid <ins class="diffchange diffchange-inline">[http://partsregistry</ins>.<ins class="diffchange diffchange-inline">org/wiki/index.php?title=Part:BBa_K283002 '''BBa_K283002'''] </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| align="center" border="1px" cellpadding="4px"</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| align="center" border="1px" cellpadding="4px"</div></td></tr>
</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Speed_Control_Methodology&diff=148580&oldid=prevYubin: /* Step 1 */2009-10-21T16:50:28Z<p><span class="autocomment">Step 1</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>YBE02 and YBS02 (delta ''cheZ'') strain were used to test whether the inducible regulation of ''cheZ'' would succeed. With no background expression of ''cheZ'', we would be able to achieve precise control over intracellular cheZ level through regulated gene expression. The plasmid <del class="diffchange diffchange-inline"> </del>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 '''BBa_K283002'''] was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of and YBE02 and YBS02 of ''cheZ'' expression was induced through incubating with different concentrations of aTc and various induced time. The samples were gathered at specific time and with different aTc concentrations as follows.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>YBE02 and YBS02 (delta ''cheZ'') strain were used to test whether the inducible regulation of ''cheZ'' would succeed. With no background expression of ''cheZ'', we would be able to achieve precise control over intracellular cheZ level through regulated gene expression. The plasmid <ins class="diffchange diffchange-inline">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K283014 '''BBa_K283014'''] or </ins>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 '''BBa_K283002'''] was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of and YBE02 and YBS02 of ''cheZ'' expression was induced through incubating with different concentrations of <ins class="diffchange diffchange-inline">IPTG or </ins>aTc and various induced time. The samples were gathered at specific time and with different <ins class="diffchange diffchange-inline">IPTG or </ins>aTc concentrations as follows.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| align="center" border="1px" cellpadding="4px"</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| align="center" border="1px" cellpadding="4px"</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>! rowspan="2" | Culture Time !! colspan="4" | aTc concentration</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>! rowspan="2" | Culture Time !! colspan="4" | <ins class="diffchange diffchange-inline">IPTG or </ins>aTc concentration</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>! 0uM !! 1uM !! 2uM !! 3.3uM</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>! 0uM !! 1uM !! 2uM !! 3.3uM</div></td></tr>
</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Speed_Control_Methodology&diff=148553&oldid=prevYubin: /* Step 3 */2009-10-21T16:48:50Z<p><span class="autocomment">Step 3</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
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<td colspan='2' style="background-color: white; color:black;">Revision as of 16:48, 21 October 2009</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 3===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 3===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>PCR was applied with test primers and with single colonies on the plate as templates to examine if the recombination strategy was successful.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>PCR was applied with test primers and with single colonies on the plate as templates to examine if the recombination strategy was successful.</div></td></tr>
<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;"></del></div></td><td colspan="2"> </td></tr>
<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;">[[Image:HKU-HKBU_speed_control_protocols_recombineering_of_cheZ.png |center|thumb|300px]]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Regulation of ''cheZ'' expression==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Regulation of ''cheZ'' expression==</div></td></tr>
</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Speed_Control_Methodology&diff=148129&oldid=prevYubin at 16:29, 21 October 20092009-10-21T16:29:39Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
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<td colspan='2' style="background-color: white; color:black;">Revision as of 16:29, 21 October 2009</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>YBE02 and YBS02 (delta ''cheZ'') strain were used to test whether the inducible regulation of ''cheZ'' would succeed. With no background expression of ''cheZ'', we would be able to achieve precise control over intracellular cheZ level through regulated gene expression. The plasmid <del class="diffchange diffchange-inline">( </del>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 BBa_K283002] <del class="diffchange diffchange-inline">) </del>was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of and YBE02 and YBS02 <del class="diffchange diffchange-inline">different </del>''cheZ'' expression <del class="diffchange diffchange-inline">level </del>was induced through incubating with different concentrations of <del class="diffchange diffchange-inline">IPTG </del>and various induced time. The samples were gathered at specific time and with different <del class="diffchange diffchange-inline">IPTG </del>concentrations as follows.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>YBE02 and YBS02 (delta ''cheZ'') strain were used to test whether the inducible regulation of ''cheZ'' would succeed. With no background expression of ''cheZ'', we would be able to achieve precise control over intracellular cheZ level through regulated gene expression. The plasmid <ins class="diffchange diffchange-inline"> </ins>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 <ins class="diffchange diffchange-inline">'''</ins>BBa_K283002<ins class="diffchange diffchange-inline">'''</ins>] <ins class="diffchange diffchange-inline"> </ins>was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of and YBE02 and YBS02 <ins class="diffchange diffchange-inline">of </ins>''cheZ'' expression was induced through incubating with different concentrations of <ins class="diffchange diffchange-inline">aTc </ins>and various induced time. The samples were gathered at specific time and with different <ins class="diffchange diffchange-inline">aTc </ins>concentrations as follows.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| align="center" border="1px" cellpadding="4px"</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| align="center" border="1px" cellpadding="4px"</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>! rowspan="2" | Culture Time !! colspan="4" | <del class="diffchange diffchange-inline">IPTG </del>concentration</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>! rowspan="2" | Culture Time !! colspan="4" | <ins class="diffchange diffchange-inline">aTc </ins>concentration</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>! 0uM !! 1uM !! 2uM !! 3.3uM</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>! 0uM !! 1uM !! 2uM !! 3.3uM</div></td></tr>
</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Speed_Control_Methodology&diff=148060&oldid=prevYubin at 16:26, 21 October 20092009-10-21T16:26:22Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 16:26, 21 October 2009</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>YBE02 and YBS02 (delta ''cheZ'') strain were used to test whether the inducible regulation of ''cheZ'' would succeed. With no background expression of ''cheZ'', we would be able to achieve precise control over intracellular cheZ level through regulated gene expression. The plasmid <del class="diffchange diffchange-inline">pIac-his-cheZ-cm </del>was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of <del class="diffchange diffchange-inline">MG3 </del>and different cheZ expression level was induced through incubating with different concentrations of IPTG and various induced time. The samples were gathered at specific time and with different IPTG concentrations as follows.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>YBE02 and YBS02 (delta ''cheZ'') strain were used to test whether the inducible regulation of ''cheZ'' would succeed. With no background expression of ''cheZ'', we would be able to achieve precise control over intracellular cheZ level through regulated gene expression. The plasmid <ins class="diffchange diffchange-inline">( [http://partsregistry.org/wiki/index.php?title=Part:BBa_K283002 BBa_K283002] ) </ins>was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of <ins class="diffchange diffchange-inline"> and YBE02 </ins>and <ins class="diffchange diffchange-inline">YBS02 </ins>different <ins class="diffchange diffchange-inline">''</ins>cheZ<ins class="diffchange diffchange-inline">'' </ins>expression level was induced through incubating with different concentrations of IPTG and various induced time. The samples were gathered at specific time and with different IPTG concentrations as follows.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| align="center" border="1px" cellpadding="4px"</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| align="center" border="1px" cellpadding="4px"</div></td></tr>
</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Speed_Control_Methodology&diff=147915&oldid=prevYubin at 16:20, 21 October 20092009-10-21T16:20:24Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Speed Control - Methodology=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Speed Control - Methodology=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>==<del class="diffchange diffchange-inline">CheZ knockout of </del>''<del class="diffchange diffchange-inline">YBE01</del>'' and <del class="diffchange diffchange-inline">''</del>YBS01<del class="diffchange diffchange-inline">''</del>==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>==''<ins class="diffchange diffchange-inline">cheZ</ins>'' <ins class="diffchange diffchange-inline">knockout of YBE01 </ins>and YBS01==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Team:HKU-HKBU/Protocols#Recombineering | Recombineering]] strategy is used for the ''cheZ'' <del class="diffchange diffchange-inline">gene of ''</del>YBE01<del class="diffchange diffchange-inline">'' </del>and <del class="diffchange diffchange-inline">''</del>YBS01<del class="diffchange diffchange-inline">''</del>. Because the protocols for the knockout process are the same for these two strains, we just took <del class="diffchange diffchange-inline">''</del>YBS01<del class="diffchange diffchange-inline">'' </del>as an example to illustrate the whole procedures.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Team:HKU-HKBU/Protocols#Recombineering | Recombineering]] strategy is used for the ''cheZ'' <ins class="diffchange diffchange-inline">knockout in </ins>YBE01 and YBS01. Because the protocols for the knockout process are the same for these two strains, we just took YBS01 as an example to illustrate the whole procedures.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>A psim6 plasmid, which could help Recombineering process, was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of <del class="diffchange diffchange-inline">''</del>YBS01<del class="diffchange diffchange-inline">'' </del>by electroporation. After recovering with SOC solution for 30 minutes, they were spread to Agar plates with Ampicillin resistance. Then one single colony on the plate was picked up for [[Team:HKU-HKBU/Protocols#Pre-culture | pre-culture]].</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>A psim6 plasmid, which could help Recombineering process, was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of YBS01 by electroporation. After recovering with SOC solution for 30 minutes, they were spread to Agar plates with Ampicillin resistance. Then one single colony on the plate was picked up for [[Team:HKU-HKBU/Protocols#Pre-culture | pre-culture]].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 2===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 2===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>A DNA fragment with homologeous arms made by PCR using specific primers was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the <del class="diffchange diffchange-inline">''</del>YBS01<del class="diffchange diffchange-inline">'' </del>with psim6. After recovering with SOC solution for <del class="diffchange diffchange-inline">30 </del>minutes, they were spread to Agar plates with <del class="diffchange diffchange-inline">Chloramphenicol </del>resistance. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>A DNA fragment with homologeous arms made by PCR using specific primers was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the YBS01 with psim6. After recovering with SOC solution for <ins class="diffchange diffchange-inline">120 </ins>minutes, they were spread to Agar plates with <ins class="diffchange diffchange-inline">chloramphenicol </ins>resistance. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 3===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 3===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>PCR was <del class="diffchange diffchange-inline">done </del>with test primers and with single colonies on the plate as templates to examine if the recombination strategy was successful.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>PCR was <ins class="diffchange diffchange-inline">applied </ins>with test primers and with single colonies on the plate as templates to examine if the recombination strategy was successful.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HKU-HKBU_speed_control_protocols_recombineering_of_cheZ.png |center|thumb|300px]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HKU-HKBU_speed_control_protocols_recombineering_of_cheZ.png |center|thumb|300px]]</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 22:</td>
<td colspan="2" class="diff-lineno">Line 22:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 1===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">MG3 </del>(delta <del class="diffchange diffchange-inline">CheZ</del>) strain <del class="diffchange diffchange-inline">was </del>used to test whether the inducible regulation of ''cheZ'' would succeed. With no background expression of cheZ, we would be able to achieve precise control over intracellular cheZ level through regulated gene expression. The plasmid pIac-his-cheZ-cm was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of MG3 and different cheZ expression level was induced through incubating with different concentrations of IPTG and various induced time. The samples were gathered at specific time and with different IPTG concentrations as follows.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">YBE02 and YBS02 </ins>(delta <ins class="diffchange diffchange-inline">''cheZ''</ins>) strain <ins class="diffchange diffchange-inline">were </ins>used to test whether the inducible regulation of ''cheZ'' would succeed. With no background expression of <ins class="diffchange diffchange-inline">''</ins>cheZ<ins class="diffchange diffchange-inline">''</ins>, we would be able to achieve precise control over intracellular cheZ level through regulated gene expression. The plasmid pIac-his-cheZ-cm was [[Team:HKU-HKBU/Protocols#Transformation | transformed]] into the [[Team:HKU-HKBU/Protocols#Competent_Cell_Preparation_for_Electro_Transformation | competent cell]] of MG3 and different cheZ expression level was induced through incubating with different concentrations of IPTG and various induced time. The samples were gathered at specific time and with different IPTG concentrations as follows.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| align="center" border="1px" cellpadding="4px"</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{| align="center" border="1px" cellpadding="4px"</div></td></tr>
</table>Yubinhttp://2009.igem.org/wiki/index.php?title=Team:HKU-HKBU/Speed_Control_Methodology&diff=128850&oldid=prevUtopian: /* Step 2 */2009-10-20T14:43:04Z<p><span class="autocomment">Step 2</span></p>
<table style="background-color: white; color:black;">
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<td colspan='2' style="background-color: white; color:black;">Revision as of 14:43, 20 October 2009</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 2===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 2===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The bacteria [[Team:HKU-HKBU/Protocols#Bacteria_Lysis | bacteria lysis]] underwent to release the protein samples. [[Team:HKU-HKBU/Protocols#BCA_Quantification_Analysis | BCA quantification analysis]] was done to detect the concentrations of these protein samples. After analyzing the concentrations of each protein samples, we used the formula: total amount of protein= sample concentration*sample volume to adjust the sample volume to the same loading volume by adding the mixture buffer. Equal loading of protein samples were ensured in [[Team:HKU-HKBU/Protocols#Western_Blotting | <del class="diffchange diffchange-inline">western </del>blotting]] after these adjustments.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The bacteria [[Team:HKU-HKBU/Protocols#Bacteria_Lysis | bacteria lysis]] underwent to release the protein samples. [[Team:HKU-HKBU/Protocols#BCA_Quantification_Analysis | BCA quantification analysis]] was done to detect the concentrations of these protein samples. After analyzing the concentrations of each protein samples, we used the formula: total amount of protein= sample concentration*sample volume to adjust the sample volume to the same loading volume by adding the mixture buffer. Equal loading of protein samples were ensured in [[Team:HKU-HKBU/Protocols#Western_Blotting | <ins class="diffchange diffchange-inline">Western </ins>blotting]] after these adjustments.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 3===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Step 3===</div></td></tr>
</table>Utopian