http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Methyl_Salicylate&feed=atom&action=historyTeam:UNITN-Trento/Project/Methyl Salicylate - Revision history2024-03-28T22:16:40ZRevision history for this page on the wikiMediaWiki 1.16.5http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Methyl_Salicylate&diff=306896&oldid=prevGgirelli at 22:55, 4 October 20132013-10-04T22:55:25Z<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 22:55, 4 October 2013</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 28:</td>
<td colspan="2" class="diff-lineno">Line 28:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-subtitle">MeSA detection</span> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-subtitle">MeSA detection</span> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> MeSA is an highly volatile liquid with a distinct minty fragrance. We exploited the physical properties of MeSA to quantify its production by gas chromatography using a Finnigan Trace GC ULTRA connected to a <b>flame ionization detector</b> (FID). This kind of instrument<del class="diffchange diffchange-inline">, </del>is able to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM). For more details about the protocol that we used for the instrument <del class="diffchange diffchange-inline">see </del><a href=" https://2013.igem.org/Team:UNITN-Trento/Protocols#MeSA-detection"> here<del class="diffchange diffchange-inline">. </del></a> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> MeSA is an highly volatile liquid with a distinct minty fragrance. We exploited the physical properties of MeSA to quantify its production by gas chromatography using a Finnigan Trace GC ULTRA connected to a <b>flame ionization detector</b> (FID). This kind of instrument is able to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM). For more details about the protocol that we used for the instrument <ins class="diffchange diffchange-inline">check </ins><a href=" https://2013.igem.org/Team:UNITN-Trento/Protocols#MeSA-detection">here</a> <ins class="diffchange diffchange-inline">.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </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 colspan="2" class="diff-lineno">Line 35:</td>
<td colspan="2" class="diff-lineno">Line 35:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_Pedro_GC.JPG" /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img class="plot" style="height: 280px; width: auto;" src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_Pedro_GC.JPG" /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <span style="text-align:justify;" class="tn-caption center"><b>Figure 3<del class="diffchange diffchange-inline">:</del></b> Left panel: calibration curve obtained with different concentrations of pure MeSA in ethanol. We chose to use ethanol to build up the calibration curve because at the beginning we had some <del class="diffchange diffchange-inline">problems </del>with the solubility of the methyl salicylate. However, during our experience <del class="diffchange diffchange-inline">were also </del>performed <del class="diffchange diffchange-inline">many measures </del>to verify the equivalence of using ethanol and LB as matrix for dissolving MeSA. Right panel: GC-FID in action </span></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <span style="text-align:justify;" class="tn-caption center"><b>Figure 3<ins class="diffchange diffchange-inline">.</ins></b> Left panel: calibration curve obtained with different concentrations of pure MeSA in ethanol. We chose to use ethanol to build up the calibration curve because at the beginning we had some <ins class="diffchange diffchange-inline">issues </ins>with the solubility of the methyl salicylate. However, during our experience <ins class="diffchange diffchange-inline">we </ins>performed <ins class="diffchange diffchange-inline">other measurements </ins>to verify the equivalence of using ethanol and LB as <ins class="diffchange diffchange-inline">a </ins>matrix for dissolving MeSA. Right panel: GC-FID in action </span></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: #ffa; color:black; font-size: smaller;"><div> Once obtained the calibration curve, NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> were grown both in LB and M9 medium<del class="diffchange diffchange-inline">, </del>induced with 5 mM arabinose and in some cases supplemented with salicylic acid. All the gas chromatography <del class="diffchange diffchange-inline">measures here </del>reported were <del class="diffchange diffchange-inline">done </del>in liquid phase, by injecting 1 <del class="diffchange diffchange-inline">ul </del>of pre-filtered culture in the instrument.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> Once obtained the calibration curve, NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> were grown both in LB and M9 medium induced with 5 mM arabinose and in some cases supplemented with salicylic acid. All the gas chromatography <ins class="diffchange diffchange-inline">measurements </ins>reported <ins class="diffchange diffchange-inline">here </ins>were <ins class="diffchange diffchange-inline">performed </ins>in liquid phase, by injecting 1 <ins class="diffchange diffchange-inline">&micro;l </ins>of pre-filtered culture in the instrument.</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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img src="https://static.igem.org/mediawiki/2013/d/d4/Induced_sample_produce_MeSA.png"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img src="https://static.igem.org/mediawiki/2013/d/d4/Induced_sample_produce_MeSA.png"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <span class="tn-caption center" style="text-align:justify;"><b>Figure 4:</b> induced sample produces MeSA. A culture of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was grown until O.D. 0.6 was reached. The culture was then splitted in 2 samples and one was induced with 5 mM arabinose. 2 mM salycilic acid was added to these samples. After about 4 h the samples were connected to the Gas Chromatograph. The induced sample (blue <del class="diffchange diffchange-inline">trace</del>) shows the characteristic peak of methyl salicylate, as opposed to non induced cells (red <del class="diffchange diffchange-inline">trace</del>).</span></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <span class="tn-caption center" style="text-align:justify;"><b>Figure 4:</b> induced sample produces MeSA. A culture of <ins class="diffchange diffchange-inline">NEB10&beta; </ins>cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was grown until O.D. 0.6 was reached. The culture was then splitted in 2 samples and one was induced with 5 mM arabinose. 2 mM salycilic acid was added to these samples. After about 4 h the samples were connected to the Gas Chromatograph. The induced sample (blue) shows the characteristic peak of methyl salicylate, as opposed to non induced cells (red).</span></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> Once we had all the chromatograms, with the software <i> Finningan Xcalibur® </i>, we were able to obtain directly the MeSA quantities from each <del class="diffchange diffchange-inline">bacteria’s </del>samples. Below we <del class="diffchange diffchange-inline">have reported </del>the most significant data.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> Once we had all the chromatograms, with the software <i> Finningan Xcalibur® </i>, we were able to obtain directly the MeSA quantities from each <ins class="diffchange diffchange-inline">bacterial </ins>samples. Below we <ins class="diffchange diffchange-inline">report </ins>the most significant data.</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> <img src="https://static.igem.org/mediawiki/2013/3/3f/Tn-2013_Istogramma_mesa.jpg"/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img src="https://static.igem.org/mediawiki/2013/3/3f/Tn-2013_Istogramma_mesa.jpg"/></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: #ffa; color:black; font-size: smaller;"><div> <span style="text-align:justify;" class="tn-caption center"><b>Figure 5:</b> <del class="diffchange diffchange-inline">Quantification </del>of MeSA by GC-FID. <del class="diffchange diffchange-inline"> </del>NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> supplemented with salycilic acid produce around 0.4 mM of MeSA. Non transformed cells and non induced cells did not produce any MeSA. Cells induced with arabinose and not supplemented with salycilic acid did not show any significant MeSA concentration (data not shown).</span></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <span style="text-align:justify;" class="tn-caption center"><b>Figure 5:</b> <ins class="diffchange diffchange-inline">quantification </ins>of MeSA by GC-FID. NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> supplemented with salycilic acid produce around 0.4 mM of MeSA. Non transformed cells and non induced cells did not produce any MeSA. Cells induced with arabinose and not supplemented with salycilic acid did not show any significant MeSA concentration (data not shown).</span></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-subtitle">MeSA: 1ppm is better than zero</span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-subtitle">MeSA: 1ppm is better than zero</span></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p>In addition to measurements in the liquid phase, we also tried to quantify the amount of MeSA produced by our device and able to escape in the gas phase.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p>In addition to measurements in the liquid phase, we also tried to quantify the amount of MeSA produced by our device and able to escape in the gas phase.</p></div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 55:</td>
<td colspan="2" class="diff-lineno">Line 56:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <center><img class="plot"style="width:49%;border: 3px solid white;</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <center><img class="plot"style="width:49%;border: 3px solid white;</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>box-shadow: 2px 2px 4px #323232;margin-top: -6px;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>box-shadow: 2px 2px 4px #323232;margin-top: -6px;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) <del class="diffchange diffchange-inline">NEB10β </del>cells transformed with <del class="diffchange diffchange-inline">Bba_K1065102 </del>and induced with <del class="diffchange diffchange-inline">5mM </del>arabinose, b) <del class="diffchange diffchange-inline">NEB10β </del>cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106"><del class="diffchange diffchange-inline">Bba_K1065106</del></a> and induced with <del class="diffchange diffchange-inline">5mM </del>arabinose and supplemented with salycilic acid, c) 15 ppm reference point.</span></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. <ins class="diffchange diffchange-inline">(</ins>a) <ins class="diffchange diffchange-inline">NEB10&beta; </ins>cells transformed with <ins class="diffchange diffchange-inline">BBa_K1065102 </ins>and induced with <ins class="diffchange diffchange-inline">5 mM </ins>arabinose, <ins class="diffchange diffchange-inline">(</ins>b) <ins class="diffchange diffchange-inline">NEB10&beta; </ins>cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106"><ins class="diffchange diffchange-inline">BBa_K1065106</ins></a> and induced with <ins class="diffchange diffchange-inline">5 mM </ins>arabinose and supplemented with salycilic acid, <ins class="diffchange diffchange-inline">(</ins>c) 15 ppm reference point.</span></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106"><del class="diffchange diffchange-inline">Bba_K1065106</del></a><del class="diffchange diffchange-inline">, </del>were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed <del class="diffchange diffchange-inline">the </del>gas <del class="diffchange diffchange-inline">chromatographyc analyis </del>with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a <del class="diffchange diffchange-inline">ritension </del>time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental <del class="diffchange diffchange-inline">condition</del>: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of salicylic acid). Non induced cells did not produce any MeSA (data not shown).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106"><ins class="diffchange diffchange-inline">BBa_K1065106</ins></a> were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed <ins class="diffchange diffchange-inline">a </ins>gas <ins class="diffchange diffchange-inline">chromatography </ins>with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a <ins class="diffchange diffchange-inline">retention </ins>time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental <ins class="diffchange diffchange-inline">conditions</ins>: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of salicylic acid). Non induced cells did not produce any MeSA (data not shown).</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p><br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p><br/></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Summary</span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Summary</span></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> Our MeSA devices <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <del class="diffchange diffchange-inline"> </del><a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a> were able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). Additionally, it seems that more MeSA is present in the liquid phase than in the gas phase. <br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> Our MeSA devices <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a> were able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). Additionally, it seems that more MeSA is present in the liquid phase than in the gas phase. <br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we <del class="diffchange diffchange-inline">realised </del>that the <del class="diffchange diffchange-inline">pLAC </del>promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we <ins class="diffchange diffchange-inline">realized </ins>that the <ins class="diffchange diffchange-inline">pLac </ins>promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </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: #eee; color:black; font-size: smaller;"><div> </div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div class="sheet-2"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div class="sheet-2"></div></td></tr>
</table>Ggirellihttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Methyl_Salicylate&diff=306296&oldid=prevGgirelli at 22:38, 4 October 20132013-10-04T22:38:54Z<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 22:38, 4 October 2013</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 10:</td>
<td colspan="2" class="diff-lineno">Line 10:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Results - Methyl Salicylate </span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Results - Methyl Salicylate </span></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <i>B. fruity</i> <del class="diffchange diffchange-inline">needed </del>also a fruit ripening <del class="diffchange diffchange-inline">ihnibitor</del>. It was difficult to find a volatile molecule that could be enzymatically produced by a bacteria and also <del class="diffchange diffchange-inline">proofed </del>to be an efficient ripening <del class="diffchange diffchange-inline">inhbitor</del>. There were not many candidates to choose from and after a long search we found methyl salicylate (MeSA). Previous <del class="diffchange diffchange-inline">work </del>suggested that MeSA inhibits the ripening of kiwifruit <span class="tn-ref"> (Aghdam M. et al., Journal of Agricultural Science. June 2011, Vol. 3, 2, pp. 149-156)</span> and tomatoes, at a concentration of 0.5 mM <span class="tn-ref">(Ding, C. and Wang, Plant Science 2003, Y. 164 pp. 589-596)</span>. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <i>B. fruity</i> <ins class="diffchange diffchange-inline">needs </ins>also a fruit ripening <ins class="diffchange diffchange-inline">inhibitor</ins>. It was difficult to find a volatile molecule that could be enzymatically produced by a bacteria and also <ins class="diffchange diffchange-inline">demonstrated </ins>to be an efficient ripening <ins class="diffchange diffchange-inline">inhibitor</ins>. There were not many candidates to choose from and after a long search we found methyl salicylate (MeSA). Previous <ins class="diffchange diffchange-inline">works </ins>suggested that MeSA inhibits the ripening of kiwifruit <span class="tn-ref"> (Aghdam M. et al., Journal of Agricultural Science. June 2011, Vol. 3, 2, pp. 149-156)</span> and tomatoes, at a concentration of 0.5 mM <span class="tn-ref">(Ding, C. and Wang, Plant Science 2003, Y. 164 pp. 589-596)</span>. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> We were happy to find out that many of the <del class="diffchange diffchange-inline">needed </del>parts to produce MeSA were already available in the registry. These parts were <del class="diffchange diffchange-inline">initally </del>built by the MIT 2006 iGEM team for the project <a href="http://openwetware.org/wiki/IGEM:MIT/2006/Blurb"><i>Eau de coli</i></a>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> We were happy to find out that many of the parts <ins class="diffchange diffchange-inline">required </ins>to produce MeSA were already available in the registry. These parts were built by the MIT 2006 iGEM team for the project <a href="http://openwetware.org/wiki/IGEM:MIT/2006/Blurb"><i>Eau de coli</i></a>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </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> <img id="mesapath" style= "margin-bottom: 1em"; src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_MeSA_path.jpg"/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img id="mesapath" style= "margin-bottom: 1em"; src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_MeSA_path.jpg"/></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> <span class="tn-caption" style="text-align:justify;"> <b> Figure 1: </b> <del class="diffchange diffchange-inline">In </del>this picture <del class="diffchange diffchange-inline">is shown </del>the pathway that was exploited to produce <del class="diffchange diffchange-inline">Methyl Salicyalte</del>. The precursor is <del class="diffchange diffchange-inline">the </del>chorismate, a metabolic intermediate of the Shikimate pathway <del class="diffchange diffchange-inline">which </del>many plants and bacteria (like <i> E.coli </i> and <i> B.subtilis </i>) <del class="diffchange diffchange-inline">have</del>. <del class="diffchange diffchange-inline">The </del>chorismate undergoes <del class="diffchange diffchange-inline">firstly </del>a reaction of isomerization by the isochorismate synthase<del class="diffchange diffchange-inline">, </del>PchA and then the salicylate is obtained by the action of PchB an isochorismate pyruvate lyase. Both enzymes are from the <del class="diffchange diffchange-inline">micro-organism </del><i>Pseudomonas aeruginosa </i>. In the final part of the reaction<del class="diffchange diffchange-inline">, </del>BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase. This enzyme is already present in <del class="diffchange diffchange-inline">the genome of </del><i>E. coli</i>. We thought that adding another copy of this gene would ultimately result in an <del class="diffchange diffchange-inline">increase of </del>MeSA production. </span></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <span class="tn-caption" style="text-align:justify;"> <b> Figure 1: </b> this picture <ins class="diffchange diffchange-inline">shows </ins>the pathway that was exploited to produce <ins class="diffchange diffchange-inline">methyl salicyalte</ins>. The precursor is chorismate, a metabolic intermediate of the Shikimate pathway <ins class="diffchange diffchange-inline">present in </ins>many plants and bacteria (like <i>E. coli</i> and <i>B. subtilis</i>). <ins class="diffchange diffchange-inline">At first, </ins>chorismate undergoes a reaction of isomerization by the isochorismate synthase <ins class="diffchange diffchange-inline">(</ins>PchA<ins class="diffchange diffchange-inline">) </ins>and then the salicylate is obtained by the action of PchB<ins class="diffchange diffchange-inline">, </ins>an isochorismate pyruvate lyase. Both enzymes are from the <ins class="diffchange diffchange-inline">microorganism </ins><i>Pseudomonas aeruginosa</i>. In the final part of the reaction BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase. This enzyme is already present in <i>E. coli</i>. We thought that adding another copy of this gene would ultimately result in an <ins class="diffchange diffchange-inline">increased </ins>MeSA production. </span></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></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> We modified and improved these parts and resubmitted them to the registry. <del class="diffchange diffchange-inline">For example</del>, we substituted the pTet promoter controlling <del class="diffchange diffchange-inline">the </del>BSMT1 <del class="diffchange diffchange-inline">enzyme </del>with <del class="diffchange diffchange-inline">an </del>araC-pBAD <del class="diffchange diffchange-inline">promoter</del>. <del class="diffchange diffchange-inline">Additionally the </del>MIT team did not include in their MeSA generator device <del class="diffchange diffchange-inline">the enzyme </del>SAM synthetase, that we hope will boost MeSA production. We also have re-submitted in pSB1C3 <del class="diffchange diffchange-inline">the </del>single <del class="diffchange diffchange-inline">enzymes </del>of the pathway.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> We modified and improved these parts and resubmitted them to the registry. <ins class="diffchange diffchange-inline">Especially</ins>, we substituted the pTet promoter controlling BSMT1 with araC-pBAD. <ins class="diffchange diffchange-inline">The </ins>MIT team did not include in their MeSA generator device SAM synthetase, that we hope will boost MeSA production. We also have re-submitted in pSB1C3 <ins class="diffchange diffchange-inline">each </ins>single <ins class="diffchange diffchange-inline">enzyme </ins>of the pathway.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </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>
</table>Ggirellihttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Methyl_Salicylate&diff=293774&oldid=prevXli at 14:26, 4 October 20132013-10-04T14:26:48Z<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 14:26, 4 October 2013</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 53:</td>
<td colspan="2" class="diff-lineno">Line 53:</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> </div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <center><img class="plot"style="width:49%;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/></center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <center><img class="plot"style="width:49%<ins class="diffchange diffchange-inline">;border: 3px solid white;</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">box-shadow: 2px 2px 4px #323232;margin-top: -6px</ins>;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/></center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) 15 ppm reference point.</span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) 15 ppm reference point.</span></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td></tr>
</table>Xlihttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Methyl_Salicylate&diff=293742&oldid=prevXli at 14:25, 4 October 20132013-10-04T14:25:01Z<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 14:25, 4 October 2013</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 49:</td>
<td colspan="2" class="diff-lineno">Line 49:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p>In addition to measurements in the liquid phase, we also tried to quantify the amount of MeSA produced by our device and able to escape in the gas phase.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p>In addition to measurements in the liquid phase, we also tried to quantify the amount of MeSA produced by our device and able to escape in the gas phase.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div class="tn-doublephoto-wrap"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div class="tn-doublephoto-wrap"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <img class="plot"style="width:<del class="diffchange diffchange-inline">32</del>%;"src="https://static.igem.org/mediawiki/2013/0/03/Tn-2013_BBa_K1065102_induced.jpg"/></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <img class="plot"style="width:<ins class="diffchange diffchange-inline">49</ins>%;"src="https://static.igem.org/mediawiki/2013/0/03/Tn-2013_BBa_K1065102_induced.jpg"/></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <img class="plot"style="width:<del class="diffchange diffchange-inline">32</del>%;"src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_BBa_K1065106_induced_%2B_SA.jpg"/></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <img class="plot"style="width:<ins class="diffchange diffchange-inline">49</ins>%;"src="https://static.igem.org/mediawiki/2013/a/a5/Tn-2013_BBa_K1065106_induced_%2B_SA.jpg"/></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> <img class="plot"style="width:32%;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </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 style="color: red; font-weight: bold; text-decoration: none;"> <center><img class="plot"style="width:49%;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/></center></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) 15 ppm reference point.</span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) 15 ppm reference point.</span></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td></tr>
</table>Xlihttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Methyl_Salicylate&diff=293681&oldid=prevCridelbianco at 14:22, 4 October 20132013-10-04T14:22:13Z<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 14:22, 4 October 2013</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 58:</td>
<td colspan="2" class="diff-lineno">Line 58:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p><br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p><br/></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Summary</span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Summary</span></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> Our MeSA devices <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a> were able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). Additionally, it seems that more MeSA is present in the liquid phase than in the gas phase. <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> Our MeSA devices <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a> were able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). Additionally, it seems that more MeSA is present in the liquid phase than in the gas phase. <ins class="diffchange diffchange-inline"><br></ins><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.</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>
</table>Cridelbiancohttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Methyl_Salicylate&diff=293672&oldid=prevCridelbianco at 14:21, 4 October 20132013-10-04T14:21:30Z<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 14:21, 4 October 2013</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 58:</td>
<td colspan="2" class="diff-lineno">Line 58:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p><br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p><br/></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Summary</span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Summary</span></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> Our MeSA <del class="diffchange diffchange-inline">device </del><a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> <del class="diffchange diffchange-inline">was </del>able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). Additionally, it seems that more MeSA is present in the liquid phase than in the gas phase. <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> Our MeSA <ins class="diffchange diffchange-inline">devices </ins><a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> <ins class="diffchange diffchange-inline">and <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a> were </ins>able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). Additionally, it seems that more MeSA is present in the liquid phase than in the gas phase. <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.</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>
</table>Cridelbiancohttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Methyl_Salicylate&diff=293575&oldid=prevCridelbianco at 14:16, 4 October 20132013-10-04T14:16:34Z<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 14:16, 4 October 2013</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 28:</td>
<td colspan="2" class="diff-lineno">Line 28:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-subtitle">MeSA detection</span> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-subtitle">MeSA detection</span> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> MeSA is an highly volatile liquid with a distinct minty fragrance. We exploited the physical properties of MeSA to quantify its production by gas chromatography using a Finnigan Trace GC ULTRA connected to a <b>flame ionization detector</b> (FID). This kind of instrument, is able to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM). For more details about the protocol that we used for the instrument see <a href=" https://2013.igem.org/Team:UNITN-Trento/Protocols#MeSA-detection"> here </a> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> MeSA is an highly volatile liquid with a distinct minty fragrance. We exploited the physical properties of MeSA to quantify its production by gas chromatography using a Finnigan Trace GC ULTRA connected to a <b>flame ionization detector</b> (FID). This kind of instrument, is able to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM). For more details about the protocol that we used for the instrument see <a href=" https://2013.igem.org/Team:UNITN-Trento/Protocols#MeSA-detection"> here<ins class="diffchange diffchange-inline">. </ins></a> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </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>
</table>Cridelbiancohttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Methyl_Salicylate&diff=292732&oldid=prevCate at 13:31, 4 October 20132013-10-04T13:31:05Z<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 13:31, 4 October 2013</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 58:</td>
<td colspan="2" class="diff-lineno">Line 58:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p><br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p><br/></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Summary</span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Summary</span></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> Our MeSA device <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). Additionally, it seems that more MeSA is present in the liquid phase than in the gas phase.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> Our MeSA device <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). Additionally, it seems that more MeSA is present in the liquid phase than in the gas phase. <ins class="diffchange diffchange-inline"><br></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.</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>
</table>Catehttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Methyl_Salicylate&diff=292722&oldid=prevCate at 13:30, 4 October 20132013-10-04T13:30:37Z<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 13:30, 4 October 2013</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 55:</td>
<td colspan="2" class="diff-lineno">Line 55:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) 15 ppm reference point.</span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) 15 ppm reference point.</span></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a>, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of salicylic acid). Non induced cells did not produce any MeSA.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a>, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of salicylic acid). Non induced cells did not produce any MeSA <ins class="diffchange diffchange-inline">(data not shown)</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p><br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p><br/></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Summary</span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Summary</span></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> Our MeSA device <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> Our MeSA device <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> was able to produce a significant concentration of MeSA only in the presence of salycilic acid. This finding was also previously observed by the MIT team in 2006 with their device (<a href="http://parts.igem.org/Part:BBa_J45700">BBa_J45700</a>). <ins class="diffchange diffchange-inline">Additionally, it seems that more MeSA is present in the liquid phase than in the gas phase.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>After we received the DNA sequencing results of the MIT part (<a href="http://parts.igem.org/Part:BBa_J45300">BBa_J45300</a>) and of our complete device (built with MIT parts) we realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.</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> </div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></div></td></tr>
</table>Catehttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Methyl_Salicylate&diff=291194&oldid=prevXli at 12:09, 4 October 20132013-10-04T12:09:36Z<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 12:09, 4 October 2013</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 53:</td>
<td colspan="2" class="diff-lineno">Line 53:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img class="plot"style="width:32%;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img class="plot"style="width:32%;"src="https://static.igem.org/mediawiki/2013/3/34/15_ppm_of_Mesa_Pure.jpg"/></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) <del class="diffchange diffchange-inline">standard curve corresponding to </del>15 ppm.</span></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <span class="tn-caption"><b>Figure 6:</b> Quantification of MeSA by gas chromatography. a) NEB10β cells transformed with Bba_K1065102 and induced with 5mM arabinose, b) NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> and induced with 5mM arabinose and supplemented with salycilic acid, c) 15 ppm <ins class="diffchange diffchange-inline">reference point</ins>.</span></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a>, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of <del class="diffchange diffchange-inline">salycilici </del>acid). Non induced cells did not produce any MeSA.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a>, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 1.3 ppm for <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_1065102</a> and 0.9 ppm for <a href="http://parts.igem.org/Part:BBa_K1065106">Bba_K1065106</a> (in the presence of <ins class="diffchange diffchange-inline">salicylic </ins>acid). Non induced cells did not produce any MeSA.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p><br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p><br/></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Summary</span></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <span class="tn-title">Summary</span></div></td></tr>
</table>Xli