http://2012.igem.org/wiki/index.php?title=Team:Calgary/Notebook/CatecholDegradation&feed=atom&action=historyTeam:Calgary/Notebook/CatecholDegradation - Revision history2024-03-28T11:13:02ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:Calgary/Notebook/CatecholDegradation&diff=263996&oldid=prevStephanie0101 at 01:35, 4 October 20122012-10-04T01:35:46Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></html>[[File:<del class="diffchange diffchange-inline">UCalgary2012ButCHgraph</del>.PNG|frame|center]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></html>[[File:<ins class="diffchange diffchange-inline">UCalgary2012ButCH_graph_(1)</ins>.PNG|frame|center]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[File:<del class="diffchange diffchange-inline">UCalgary2012Toluenegraph</del>.PNG|frame|center]] </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[File:<ins class="diffchange diffchange-inline">UCalgary2012Toluene_graph_(1)</ins>.PNG|frame|center]] </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:UCalgary2012Week9gelAHS.PNG|500px|thumb|Fig.3 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies 1-5 that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top band in lane 6 indicated successful amplification of the alkane hydroxylase system (2932 bp).|center]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:UCalgary2012Week9gelAHS.PNG|500px|thumb|Fig.3 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies 1-5 that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top band in lane 6 indicated successful amplification of the alkane hydroxylase system (2932 bp).|center]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[File:<del class="diffchange diffchange-inline">UCalgary2012Naphthalenegraph</del>.PNG|frame|center]]<html></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[File:<ins class="diffchange diffchange-inline">UCalgary2012Naphthalene_graph_(1)</ins>.PNG|frame|center]]<html></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 10 (July 2-July 6)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 10 (July 2-July 6)</h2></div></td></tr>
</table>Stephanie0101http://2012.igem.org/wiki/index.php?title=Team:Calgary/Notebook/CatecholDegradation&diff=255183&oldid=prevStephanie0101 at 06:12, 3 October 20122012-10-03T06:12:58Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></html>[[File:UCalgary2012ButCHgraph.PNG|frame|center]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></html>[[File:UCalgary2012ButCHgraph.PNG|frame|center]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[File:<del class="diffchange diffchange-inline">UCalgary2012_Toluene_assay</del>.<del class="diffchange diffchange-inline">png</del>|frame|center]] </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[File:<ins class="diffchange diffchange-inline">UCalgary2012Toluenegraph</ins>.<ins class="diffchange diffchange-inline">PNG</ins>|frame|center]] </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:UCalgary2012Week9gelAHS.PNG|500px|thumb|Fig.3 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies 1-5 that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top band in lane 6 indicated successful amplification of the alkane hydroxylase system (2932 bp).|center]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:UCalgary2012Week9gelAHS.PNG|500px|thumb|Fig.3 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies 1-5 that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top band in lane 6 indicated successful amplification of the alkane hydroxylase system (2932 bp).|center]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[File:<del class="diffchange diffchange-inline">UCalgary2012Naphthalene_assay</del>.<del class="diffchange diffchange-inline">png</del>|frame|center]]<html></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[File:<ins class="diffchange diffchange-inline">UCalgary2012Naphthalenegraph</ins>.<ins class="diffchange diffchange-inline">PNG</ins>|frame|center]]<html></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 10 (July 2-July 6)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 10 (July 2-July 6)</h2></div></td></tr>
</table>Stephanie0101http://2012.igem.org/wiki/index.php?title=Team:Calgary/Notebook/CatecholDegradation&diff=255157&oldid=prevStephanie0101 at 06:10, 3 October 20122012-10-03T06:10:32Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></html>[[File:<del class="diffchange diffchange-inline">UCalgary2012_Butchgraph</del>.<del class="diffchange diffchange-inline">png</del>|frame|center]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></html>[[File:<ins class="diffchange diffchange-inline">UCalgary2012ButCHgraph</ins>.<ins class="diffchange diffchange-inline">PNG</ins>|frame|center]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:UCalgary2012_Toluene_assay.png|frame|center]] </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:UCalgary2012_Toluene_assay.png|frame|center]] </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:UCalgary2012Week9gelAHS.PNG|500px|thumb|Fig.3 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies 1-5 that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top band in lane 6 indicated successful amplification of the alkane hydroxylase system (2932 bp).|center]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:UCalgary2012Week9gelAHS.PNG|500px|thumb|Fig.3 Alkane Hydroxylase system verification colony PCR gel. The alkane hydroxylase system (K398014) was found in the 2012 parts registry and was transformed into top 10 E.coli cells. Lane 1 contains a 1Kb plus ladder, lanes 2-6 contain colonies 1-5 that grew on the transformation plate, lanes 7 and 8 contain the positive (RFP) and negative controls and lane 9 contains a 1Kb plus ladder. The top band in lane 6 indicated successful amplification of the alkane hydroxylase system (2932 bp).|center]]</div></td></tr>
</table>Stephanie0101http://2012.igem.org/wiki/index.php?title=Team:Calgary/Notebook/CatecholDegradation&diff=254039&oldid=prevStephanie0101 at 04:11, 3 October 20122012-10-03T04:11:30Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The Catechol assay was repeated with a new procedure. Overnight cultures were made in LB and washed the pellet in M9-MM without glucose for varying amounts of time. These cultures were spun down and the supernatant was brought to a concentration of 0.1 M. All of the supernatants became bright yellow except for the control which did not contain the <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. The colour change indicated that catechol was being converted to 2-HMS (2-Hydroxymuconate semialdehyde). The three way ligation didn’t work so a two-way ligation with <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> (tetR) in a <a href=http://partsregistry.org/Part:pSB1A3>pSB1A3</a> vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i>) in a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. One reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the insert, and another reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the insert and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the vector. Both ways worked and the new procedure for the catechol assay was done with the transformed cells and all of the supernatants turned bright yellow.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The Catechol assay was repeated with a new procedure. Overnight cultures were made in LB and washed the pellet in M9-MM without glucose for varying amounts of time. These cultures were spun down and the supernatant was brought to a concentration of 0.1 M. All of the supernatants became bright yellow except for the control which did not contain the <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. The colour change indicated that catechol was being converted to 2-HMS (2-Hydroxymuconate semialdehyde). The three way ligation didn’t work so a two-way ligation with <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> (tetR) in a <a href=http://partsregistry.org/Part:pSB1A3>pSB1A3</a> vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i>) in a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. One reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the insert, and another reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the insert and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the vector. Both ways worked and the new procedure for the catechol assay was done with the transformed cells and all of the supernatants turned bright yellow.</p></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></html>[[<del class="diffchange diffchange-inline">https://2012.igem.org/</del>File:UCalgary2012_Catechol_assay.jpg|500px|thumb||center]]<html></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|<ins class="diffchange diffchange-inline">Fig.10 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added.</ins>|center]]<html></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 style="color: red; font-weight: bold; text-decoration: none;">Fig.10 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added.</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><h2>Week 18 (August 27 - August 31)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 18 (August 27 - August 31)</h2></div></td></tr>
</table>Stephanie0101http://2012.igem.org/wiki/index.php?title=Team:Calgary/Notebook/CatecholDegradation&diff=254025&oldid=prevStephanie0101 at 04:10, 3 October 20122012-10-03T04:10:21Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The Catechol assay was repeated with a new procedure. Overnight cultures were made in LB and washed the pellet in M9-MM without glucose for varying amounts of time. These cultures were spun down and the supernatant was brought to a concentration of 0.1 M. All of the supernatants became bright yellow except for the control which did not contain the <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. The colour change indicated that catechol was being converted to 2-HMS (2-Hydroxymuconate semialdehyde). The three way ligation didn’t work so a two-way ligation with <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> (tetR) in a <a href=http://partsregistry.org/Part:pSB1A3>pSB1A3</a> vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i>) in a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. One reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the insert, and another reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the insert and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the vector. Both ways worked and the new procedure for the catechol assay was done with the transformed cells and all of the supernatants turned bright yellow.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The Catechol assay was repeated with a new procedure. Overnight cultures were made in LB and washed the pellet in M9-MM without glucose for varying amounts of time. These cultures were spun down and the supernatant was brought to a concentration of 0.1 M. All of the supernatants became bright yellow except for the control which did not contain the <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. The colour change indicated that catechol was being converted to 2-HMS (2-Hydroxymuconate semialdehyde). The three way ligation didn’t work so a two-way ligation with <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> (tetR) in a <a href=http://partsregistry.org/Part:pSB1A3>pSB1A3</a> vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i>) in a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. One reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the insert, and another reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the insert and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the vector. Both ways worked and the new procedure for the catechol assay was done with the transformed cells and all of the supernatants turned bright yellow.</p></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></html>[[https://2012.igem.org/File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.10 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added.<del class="diffchange diffchange-inline">|center]]<html></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></html>[[https://2012.igem.org/File:UCalgary2012_Catechol_assay.jpg|500px|thumb|<ins class="diffchange diffchange-inline">|center]]<html></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Fig.10 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 18 (August 27 - August 31)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 18 (August 27 - August 31)</h2></div></td></tr>
</table>Stephanie0101http://2012.igem.org/wiki/index.php?title=Team:Calgary/Notebook/CatecholDegradation&diff=254003&oldid=prevStephanie0101 at 04:08, 3 October 20122012-10-03T04:08:11Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The Catechol assay was repeated with a new procedure. Overnight cultures were made in LB and washed the pellet in M9-MM without glucose for varying amounts of time. These cultures were spun down and the supernatant was brought to a concentration of 0.1 M. All of the supernatants became bright yellow except for the control which did not contain the <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. The colour change indicated that catechol was being converted to 2-HMS (2-Hydroxymuconate semialdehyde). The three way ligation didn’t work so a two-way ligation with <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> (tetR) in a <a href=http://partsregistry.org/Part:pSB1A3>pSB1A3</a> vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i>) in a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. One reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the insert, and another reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the insert and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the vector. Both ways worked and the new procedure for the catechol assay was done with the transformed cells and all of the supernatants turned bright yellow.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The Catechol assay was repeated with a new procedure. Overnight cultures were made in LB and washed the pellet in M9-MM without glucose for varying amounts of time. These cultures were spun down and the supernatant was brought to a concentration of 0.1 M. All of the supernatants became bright yellow except for the control which did not contain the <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. The colour change indicated that catechol was being converted to 2-HMS (2-Hydroxymuconate semialdehyde). The three way ligation didn’t work so a two-way ligation with <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> (tetR) in a <a href=http://partsregistry.org/Part:pSB1A3>pSB1A3</a> vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> (<i>XylE</i>) in a <a href=http://partsregistry.org/Part:pSB1C3>pSB1C3</a> vector. One reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the vector and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the insert, and another reaction was done where <a href= http://partsregistry.org/Part:BBa_R0040>R0040</a> was the insert and <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> was the vector. Both ways worked and the new procedure for the catechol assay was done with the transformed cells and all of the supernatants turned bright yellow.</p></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;"></html>[[https://2012.igem.org/File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.10 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added.|center]]<html></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 18 (August 27 - August 31)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 18 (August 27 - August 31)</h2></div></td></tr>
</table>Stephanie0101http://2012.igem.org/wiki/index.php?title=Team:Calgary/Notebook/CatecholDegradation&diff=253627&oldid=prevStephanie0101 at 03:18, 3 October 20122012-10-03T03:18:05Z<p></p>
<table style="background-color: white; color:black;">
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<td colspan='2' style="background-color: white; color:black;">Revision as of 03:18, 3 October 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 8 (June 18 - June 22)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 8 (June 18 - June 22)</h2></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><p>This week we began a new growth assay of Pf-5, but instead of incubating it with butylcyclohexane, we incubated with naphthalene. Since naphthalene is not volatile, we no longer needed to sample the culture headspace but could directly sample remaining naphthalene in the culture itself. Like the previous experiment, we needed to establish rate of growth in M9-MM without glucose and with naphthalene as the sole carbon source. A spectrophotometer was used to measure bacterial growth. <del class="diffchange diffchange-inline"></p></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>This week we began a new growth assay of Pf-5, but instead of incubating it with butylcyclohexane, we incubated with naphthalene. Since naphthalene is not volatile, we no longer needed to sample the culture headspace but could directly sample remaining naphthalene in the culture itself. Like the previous experiment, we needed to establish rate of growth in M9-MM without glucose and with naphthalene as the sole carbon source. A spectrophotometer was used to measure bacterial growth. The original cultures were grown in LB from a Pf-5 culture in LB and subcultured into M9-MM without glucose. After 24 hours of growth these were subcultured into fresh M9-MM without glucose, again to prevent carryover of glucose from the LB. Positive controls were also made that consisted of M9-MM with glucose and Pf-5. Spectrophotometer readings of the subcultures were taken over a period of 8 days.<ins class="diffchange diffchange-inline"></p></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><p></del>The original cultures were grown in LB from a Pf-5 culture in LB and subcultured into M9-MM without glucose. After 24 hours of growth these were subcultured into fresh M9-MM without glucose, again to prevent carryover of glucose from the LB. Positive controls were also made that consisted of M9-MM with glucose and Pf-5. Spectrophotometer readings of the subcultures were taken over a period of 8 days. GC<del class="diffchange diffchange-inline">-MS </del>readings were taken of the Pf-5 incubated with butylcyclohexane after 11 days and with toluene after 10 days. The trend remained the same as the measurements taken after 5 and 6 days in the previous week. The amount of compound decreased in the experimental cultures but the amount of compound also decreased in the abiotic standards almost in equal proportions. With the results after 10 and 11 days we could see that the decrease was not due to the bacteria and we decided that the strain we were using did not contain the genes necessary for butylcyclohexane or toluene degradation.</p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p></ins>GC readings were taken of the Pf-5 incubated with butylcyclohexane after 11 days and with toluene after 10 days. The trend remained the same as the measurements taken after 5 and 6 days in the previous week. The amount of compound decreased in the experimental cultures but the amount of compound also decreased in the abiotic standards almost in equal proportions. With the results after 10 and 11 days we could see that the decrease was not due to the bacteria and we decided that the strain we were using did not contain the genes necessary for butylcyclohexane or toluene degradation.</p> </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><p>Throughout the week we continued the verification of <i>XylE</i> by performing a second colony PCR from the transformation plate using part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. Again, the gel did not contain DNA of the right lengths and only the positive control worked. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Throughout the week we continued the verification of <i>XylE</i> by performing a second colony PCR from the transformation plate using part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. Again, the gel did not contain DNA of the right lengths and only the positive control worked. </p></div></td></tr>
</table>Stephanie0101http://2012.igem.org/wiki/index.php?title=Team:Calgary/Notebook/CatecholDegradation&diff=250811&oldid=prevMaggieRY at 17:44, 2 October 20122012-10-02T17:44:40Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 8 (June 18 - June 22)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 8 (June 18 - June 22)</h2></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><p>This week we <del class="diffchange diffchange-inline">started </del>a new assay <del class="diffchange diffchange-inline">where </del>Pf-5 <del class="diffchange diffchange-inline">was </del>incubated with naphthalene. Since naphthalene is not volatile, <del class="diffchange diffchange-inline">GC </del>headspace <del class="diffchange diffchange-inline">reading was not an appropriate method of measurement </del> to <del class="diffchange diffchange-inline">indicate decrease </del>of the <del class="diffchange diffchange-inline">compound</del>. <del class="diffchange diffchange-inline">Instead we took OD600 readings </del>to measure bacterial growth. The original cultures were <del class="diffchange diffchange-inline">taken </del>from a Pf-5 culture in LB and <del class="diffchange diffchange-inline">inoculated </del>into M9-MM without glucose. After 24 hours of growth these were subcultured into fresh M9-MM without glucose, again to prevent carryover of glucose from the LB. Positive controls were also made that consisted of M9-MM with glucose and Pf-5. <del class="diffchange diffchange-inline">OD600 </del>readings of the subcultures were taken over a period of 8 days. GC readings were <del class="diffchange diffchange-inline">done on </del>the Pf-5 incubated with butylcyclohexane after 11 days and with toluene after 10 days. The trend remained the same as the measurements <del class="diffchange diffchange-inline">done </del>after 5 and 6 days in the previous week. The amount of compound decreased in the experimental cultures but the amount of compound also decreased in the abiotic standards almost in equal proportions. With the results after 10 and 11 days we could see that the decrease was not due to the bacteria and we decided that the strain we were using did not contain the genes necessary for butylcyclohexane or toluene degradation. Throughout the week we continued the verification of <i>XylE</i> by performing a second colony PCR from the transformation plate using part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. <del class="diffchange diffchange-inline">The </del>gel did not contain DNA of the right lengths and only the positive control worked. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>This week we <ins class="diffchange diffchange-inline">began </ins>a new <ins class="diffchange diffchange-inline">growth </ins>assay <ins class="diffchange diffchange-inline">of </ins>Pf-5<ins class="diffchange diffchange-inline">, but instead of incubating it with butylcyclohexane, we </ins>incubated with naphthalene. Since naphthalene is not volatile, <ins class="diffchange diffchange-inline">we no longer needed to sample the culture </ins>headspace <ins class="diffchange diffchange-inline">but could directly sample remaining naphthalene in the culture itself. </ins> <ins class="diffchange diffchange-inline">Like the previous experiment, we needed </ins>to <ins class="diffchange diffchange-inline">establish rate </ins>of <ins class="diffchange diffchange-inline">growth in M9-MM without glucose and with naphthalene as </ins>the <ins class="diffchange diffchange-inline">sole carbon source</ins>. <ins class="diffchange diffchange-inline">A spectrophotometer was used </ins>to measure bacterial growth. <ins class="diffchange diffchange-inline"></p></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 class="diffchange diffchange-inline"><p></ins>The original cultures were <ins class="diffchange diffchange-inline">grown in LB </ins>from a Pf-5 culture in LB and <ins class="diffchange diffchange-inline">subcultured </ins>into M9-MM without glucose. After 24 hours of growth these were subcultured into fresh M9-MM without glucose, again to prevent carryover of glucose from the LB. Positive controls were also made that consisted of M9-MM with glucose and Pf-5. <ins class="diffchange diffchange-inline">Spectrophotometer </ins>readings of the subcultures were taken over a period of 8 days. GC<ins class="diffchange diffchange-inline">-MS </ins>readings were <ins class="diffchange diffchange-inline">taken of </ins>the Pf-5 incubated with butylcyclohexane after 11 days and with toluene after 10 days. The trend remained the same as the measurements <ins class="diffchange diffchange-inline">taken </ins>after 5 and 6 days in the previous week. The amount of compound decreased in the experimental cultures but the amount of compound also decreased in the abiotic standards almost in equal proportions. With the results after 10 and 11 days we could see that the decrease was not due to the bacteria and we decided that the strain we were using did not contain the genes necessary for butylcyclohexane or toluene degradation.<ins class="diffchange diffchange-inline"></p> </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p></ins>Throughout the week we continued the verification of <i>XylE</i> by performing a second colony PCR from the transformation plate using part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>. <ins class="diffchange diffchange-inline">Again, the </ins>gel did not contain DNA of the right lengths and only the positive control worked. </p></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><h2>Week 9 (June 25 - June 29)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 9 (June 25 - June 29)</h2></div></td></tr>
</table>MaggieRYhttp://2012.igem.org/wiki/index.php?title=Team:Calgary/Notebook/CatecholDegradation&diff=250762&oldid=prevMaggieRY at 17:26, 2 October 20122012-10-02T17:26:30Z<p></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 17:26, 2 October 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 6 (June 4 - June 8)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 6 (June 4 - June 8)</h2></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><p> This week the experiment of testing survival and growth was repeated with the intention of determining if butylcyclohexane could be metabolized by the cells. To conclude this, the cells needed to be cultured without glucose. We subcultured from Pf-5 in M9-MM into M9-MM without glucose to avoid carry-over <del class="diffchange diffchange-inline">of glucose </del>and ensure that the bacteria use butylcyclohexane as their only carbon source. From these cultures we also obtained GC-MS (Gas Chromatography Mass Spectroscopy) readings and compared experimental values of the compound to standards made with water and butylcyclohexane. The Pf-5 samples demonstrated a decrease in amount of butylcyclohexane in the headspace (area above the liquid culture) compared to standards, indicating that the compound was being degraded by Pf-5. Furthermore, we made subcultures from original Pf-5 in M9-MM without glucose into M9-MM and toluene to test for pathways that degrade aromatic structures. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> This week the experiment of testing survival and growth was repeated with the intention of determining if butylcyclohexane could be metabolized by the cells. To conclude this, the cells needed to be cultured without glucose. We subcultured from Pf-5 in M9-MM into M9-MM without glucose to avoid carry-over and ensure that the bacteria use butylcyclohexane as their only carbon source. From these cultures we also obtained GC-MS (Gas Chromatography Mass Spectroscopy) readings and compared experimental values of the compound to standards made with water and butylcyclohexane. The Pf-5 samples demonstrated a decrease in amount of butylcyclohexane in the headspace (area above the liquid culture) compared to standards, indicating that the compound was being degraded by Pf-5. Furthermore, we made subcultures from original Pf-5 in M9-MM without glucose into M9-MM and toluene to test for pathways that degrade aromatic structures. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>We also wanted to identify which genes of Pf-5 that were part of the pathway to degrade toluene and butylcycohexane. We started running blasts on the Pf-5 genome looking for genes that are homologous to ones that we have been looking at for the two ring cleavage pathways (aromatic and aliphatic). In addition we started a verification of <i>XylE</i> (aromatic ring cleavage enzyme) by transforming part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) from the parts registry. We performed two colony PCRs. Unfortunately none of them showed successful results and only the positive control showed an appropriate length DNA. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>We also wanted to identify which genes of Pf-5 that were part of the pathway to degrade toluene and butylcycohexane. We started running blasts on the Pf-5 genome looking for genes that are homologous to ones that we have been looking at for the two ring cleavage pathways (aromatic and aliphatic). In addition we started a verification of <i>XylE</i> (aromatic ring cleavage enzyme) by transforming part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) from the parts registry. We performed two colony PCRs. Unfortunately none of them showed successful results and only the positive control showed an appropriate length DNA. </p></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><h2>Week 7 (June 11 - June 15)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 7 (June 11 - June 15)</h2></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><p> <del class="diffchange diffchange-inline">Since last week we continued taking GC readings on </del>the Pf-5 <del class="diffchange diffchange-inline">incubated with </del>butylcyclohexane and toluene. At this point the Pf-5 had been incubated with butylcyclohexane for 6 days and with toluene for 5 days. The amount of both butylcyclohexane and toluene decreased by a large amount in the experimental cultures but the amount of each in the abiotic standards also largely decreased by about the same amount. This lead us to conclude that the reason for decrease was not due to the bacteria metabolizing the compound, but due to unknown abiotic factors. To measure relative amounts of bacterial growth we <del class="diffchange diffchange-inline">took </del>OD600 <del class="diffchange diffchange-inline">readings </del>for both cultures <del class="diffchange diffchange-inline">which were </del>very low <del class="diffchange diffchange-inline">and indicated that there was </del>little bacterial growth. The verification of <i>XylE</i> from the parts registry <del class="diffchange diffchange-inline">was </del>continued <del class="diffchange diffchange-inline">using a miniprep on three </del>overnight cultures from part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. <del class="diffchange diffchange-inline">The </del>DNA concentrations were very high likely due to genomic contamination of the miniprep. <del class="diffchange diffchange-inline">A </del>second part (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) containing <i>XylE</i> and an rbs site was transformed and a colony PCR was <del class="diffchange diffchange-inline">done </del>on part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a> <del class="diffchange diffchange-inline">but the </del>gel did not indicate successful results, <del class="diffchange diffchange-inline">and </del>again only the positive control <del class="diffchange diffchange-inline">worked</del>. <del class="diffchange diffchange-inline">No positive colonies were found.</del></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> <ins class="diffchange diffchange-inline">Continuing with </ins>the <ins class="diffchange diffchange-inline">growth assays of </ins>Pf-5 <ins class="diffchange diffchange-inline">in </ins>butylcyclohexane and toluene<ins class="diffchange diffchange-inline">, we obtained more GC-MS readings</ins>. At this point the Pf-5 had been incubated with butylcyclohexane for 6 days and with toluene for 5 days. The amount of both butylcyclohexane and toluene decreased by a large amount in the experimental cultures but the amount of each in the abiotic standards also largely decreased by about the same amount. This lead us to conclude that the reason for decrease was not due to the bacteria metabolizing the compound, but due to unknown abiotic factors. To measure relative amounts of bacterial growth <ins class="diffchange diffchange-inline">in these cultures </ins>we <ins class="diffchange diffchange-inline">used a spectrophotometer at </ins>OD600 <ins class="diffchange diffchange-inline">to read absorbance </ins>for both cultures<ins class="diffchange diffchange-inline">. Optical density for both cultures was </ins>very low <ins class="diffchange diffchange-inline">indicating very </ins>little bacterial growth. <ins class="diffchange diffchange-inline"></p></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 class="diffchange diffchange-inline"><p></ins>The verification of <i>XylE</i> from the parts registry <ins class="diffchange diffchange-inline">also </ins>continued<ins class="diffchange diffchange-inline">. Three </ins>overnight cultures <ins class="diffchange diffchange-inline">were miniprepped </ins>from part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>. <ins class="diffchange diffchange-inline">Unfortunately, the </ins>DNA concentrations were very high<ins class="diffchange diffchange-inline">, </ins>likely due to genomic contamination of the miniprep. <ins class="diffchange diffchange-inline">Also, a </ins>second part (<a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>) containing <i>XylE</i> and an rbs site was transformed and a colony PCR was <ins class="diffchange diffchange-inline">performed </ins>on part <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a><ins class="diffchange diffchange-inline">. The </ins>gel did not indicate successful results, <ins class="diffchange diffchange-inline">as </ins>again<ins class="diffchange diffchange-inline">, </ins>only the positive control <ins class="diffchange diffchange-inline">PCR</ins>. </p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 8 (June 18 - June 22)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 8 (June 18 - June 22)</h2></div></td></tr>
</table>MaggieRYhttp://2012.igem.org/wiki/index.php?title=Team:Calgary/Notebook/CatecholDegradation&diff=250721&oldid=prevMaggieRY at 17:11, 2 October 20122012-10-02T17:11:38Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 6 (June 4 - June 8)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 6 (June 4 - June 8)</h2></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p> This week <del class="diffchange diffchange-inline">we made subcultures </del>from Pf-5 in M9-MM into M9-MM without glucose to avoid carry-over of glucose<del class="diffchange diffchange-inline">, to insure </del>that the bacteria use butylcyclohexane as their only carbon source. <del class="diffchange diffchange-inline">We </del>also <del class="diffchange diffchange-inline">took </del>GC (Gas Chromatography) readings and compared experimental values to standards made with water and butylcyclohexane. <del class="diffchange diffchange-inline">Readings showed </del>a decrease in amount of butylcyclohexane in the headspace<del class="diffchange diffchange-inline">, </del>compared to standards, indicating that the compound was being degraded by Pf-5<del class="diffchange diffchange-inline">. From these cultures we subcultured Pf-5 in M9-MM without glucose into the same media and butylcyclohexane, and marked this reading as our zero time point</del>. Furthermore, we made subcultures from original Pf-5 in M9-MM without glucose into M9-MM and toluene to test for pathways that degrade aromatic structures. <del class="diffchange diffchange-inline">To </del>identify <del class="diffchange diffchange-inline">possible </del>genes <del class="diffchange diffchange-inline">for </del>the <del class="diffchange diffchange-inline">pathways we </del>started running blasts on the Pf-5 genome looking for genes that are homologous to ones that we have been looking at for the two ring cleavage pathways (aromatic and aliphatic). In addition we started a verification of <i>XylE</i> (aromatic ring cleavage enzyme) by transforming part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) from the parts registry <del class="diffchange diffchange-inline">and did </del>two colony PCRs. Unfortunately none of them showed successful results and only the positive control showed an appropriate length DNA. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> This week <ins class="diffchange diffchange-inline">the experiment of testing survival and growth was repeated with the intention of determining if butylcyclohexane could be metabolized by the cells. To conclude this, the cells needed to be cultured without glucose. We subcultured </ins>from Pf-5 in M9-MM into M9-MM without glucose to avoid carry-over of glucose <ins class="diffchange diffchange-inline">and ensure </ins>that the bacteria use butylcyclohexane as their only carbon source. <ins class="diffchange diffchange-inline">From these cultures we </ins>also <ins class="diffchange diffchange-inline">obtained </ins>GC<ins class="diffchange diffchange-inline">-MS </ins>(Gas Chromatography <ins class="diffchange diffchange-inline">Mass Spectroscopy</ins>) readings and compared experimental values <ins class="diffchange diffchange-inline">of the compound </ins>to standards made with water and butylcyclohexane. <ins class="diffchange diffchange-inline">The Pf-5 samples demonstrated </ins>a decrease in amount of butylcyclohexane in the headspace <ins class="diffchange diffchange-inline">(area above the liquid culture) </ins>compared to standards, indicating that the compound was being degraded by Pf-5. Furthermore, we made subcultures from original Pf-5 in M9-MM without glucose into M9-MM and toluene to test for pathways that degrade aromatic structures. <ins class="diffchange diffchange-inline"></p></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 class="diffchange diffchange-inline"><p>We also wanted to </ins>identify <ins class="diffchange diffchange-inline">which </ins>genes <ins class="diffchange diffchange-inline">of Pf-5 that were part of </ins>the <ins class="diffchange diffchange-inline">pathway to degrade toluene and butylcycohexane. We </ins>started running blasts on the Pf-5 genome looking for genes that are homologous to ones that we have been looking at for the two ring cleavage pathways (aromatic and aliphatic). In addition we started a verification of <i>XylE</i> (aromatic ring cleavage enzyme) by transforming part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> (<i>XylE</i> with a Pcst promoter) from the parts registry<ins class="diffchange diffchange-inline">. We performed </ins>two colony PCRs. Unfortunately none of them showed successful results and only the positive control showed an appropriate length DNA. </p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 7 (June 11 - June 15)</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>Week 7 (June 11 - June 15)</h2></div></td></tr>
</table>MaggieRY