http://2010.igem.org/wiki/index.php?title=Team:BIOTEC_Dresden/ACP&feed=atom&action=historyTeam:BIOTEC Dresden/ACP - Revision history2024-03-28T15:47:07ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Team:BIOTEC_Dresden/ACP&diff=206381&oldid=prevVyctoryo at 03:00, 28 October 20102010-10-28T03:00:17Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> Although this approach isn’t quantitative, it would give a hint about the efficiency of the reaction. </p> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> Although this approach isn’t quantitative, it would give a hint about the efficiency of the reaction. </p> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p> An intermediary step for the detection of the acyl-ACP synthesis is the incubation of the reaction mixture with cell extracts of strains constitutively expressing LuxI. Using as a control LuxI extract incubated with SAM, a receiver strain and no reaction mixture, normalization is possible and the relation of fluorescence to varying amounts of the provided reaction mix containing hexanoyl-ACP can be tracked. </p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> An intermediary step for the detection of the acyl-ACP synthesis is the incubation of the reaction mixture with cell extracts of strains constitutively expressing LuxI. Using as a control LuxI extract incubated with SAM, a receiver strain and no reaction mixture, normalization is possible and the relation of fluorescence to varying amounts of the provided reaction mix containing hexanoyl-ACP can be tracked. <ins class="diffchange diffchange-inline"> Quantification can be done to assess the maximally efficient conditions for acyl-ACP synthesis. </p> </ins> </p> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p> A next integrating approach is to test our system with that of the “Fusion protein”. If we test our AHL-sensitive reporter cells again with SAM, hexanoyl-ACP containing reaction mixture and the recombinant LuxI, then the activation of the reporter cells by this reaction mixture proves the fact that we have produced both functional acyl-ACP and preserved the AHL producing function of the fusion protein<del class="diffchange diffchange-inline">. More than this, quantification can be done to assess the maximally efficient conditions for acyl</del>-<del class="diffchange diffchange-inline">ACP synthesis </del></<del class="diffchange diffchange-inline">p</del>> <del class="diffchange diffchange-inline"> </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> A next integrating approach is to test our system with that of the “Fusion protein”. If we test our AHL-sensitive reporter cells again with SAM, hexanoyl-ACP containing reaction mixture and the recombinant LuxI, then the activation of the reporter cells by this reaction mixture proves the fact that we have produced both functional acyl-ACP and preserved the AHL producing function of the fusion protein <ins class="diffchange diffchange-inline">(<i>in</ins>-<ins class="diffchange diffchange-inline">vitro</ins></<ins class="diffchange diffchange-inline">i</ins>> <ins class="diffchange diffchange-inline">activity proved). </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> Methods </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2> Methods </h2></div></td></tr>
</table>Vyctoryohttp://2010.igem.org/wiki/index.php?title=Team:BIOTEC_Dresden/ACP&diff=205581&oldid=prevLucas.schirmer at 02:31, 28 October 20102010-10-28T02:31:14Z<p></p>
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</table>Lucas.schirmerhttp://2010.igem.org/wiki/index.php?title=Team:BIOTEC_Dresden/ACP&diff=205444&oldid=prevVyctoryo at 02:24, 28 October 20102010-10-28T02:24:34Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2> Detection </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2> Detection </h2></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/2/2a/Gel222222.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/2/2a/Gel222222.jpg" class="border thumb right"></a></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/2/2a/Gel222222.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/2/2a/Gel222222.jpg" class="border thumb right"></a></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p> A first simple step to judge if the reaction is successful would be to run the reaction mixture on a gel. There is evidence that, in spite of a bigger mass, acylated ACP can travel faster than apo or holo ACP in an SDS-PAGE, due to active binding of the SDS detergent molecules to the hydrophobic acyl chain which increases the charge to mass ratio of the entire molecule, thus, facilitating faster migration (figure on the right) (2,7). On the other hand it is possible to run the reaction mixture also on a native gel and compare the migration pattern of the proteins. The potein that would migrate faster than apoACP in SDS PAGE but slower than it in a native page (separation by mass) would be the acylated ACP. Supposing that just a fraction of the ACP interacts to produce the acylated derivative, bands for both hexanoyl-ACP and apo/ holoACP were also expected. Dimerization of holo-ACP would also generate a band) </p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p> A first simple step to judge if the reaction is successful would be to run the reaction mixture on a gel. There is evidence that, in spite of a bigger mass, acylated ACP can travel faster than apo or holo ACP in an SDS-PAGE, due to active binding of the SDS detergent molecules to the hydrophobic acyl chain which increases the charge to mass ratio of the entire molecule, thus, facilitating faster migration (figure on the right<ins class="diffchange diffchange-inline">, length of acyl chains is indicated below, "-" stands for holo ACP, 6:0 - for hexanoyl-ACP</ins>) (2,7). On the other hand it is possible to run the reaction mixture also on a native gel and compare the migration pattern of the proteins. The potein that would migrate faster than apoACP in SDS PAGE but slower than it in a native page (separation by mass) would be the acylated ACP. Supposing that just a fraction of the ACP interacts to produce the acylated derivative, bands for both hexanoyl-ACP and apo/ holoACP were also expected. Dimerization of holo-ACP would also generate a band) </p> </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> Although this approach isn’t quantitative, it would give a hint about the efficiency of the reaction. </p> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> Although this approach isn’t quantitative, it would give a hint about the efficiency of the reaction. </p> </div></td></tr>
</table>Vyctoryohttp://2010.igem.org/wiki/index.php?title=Team:BIOTEC_Dresden/ACP&diff=204296&oldid=prevVyctoryo at 01:39, 28 October 20102010-10-28T01:39:31Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p> If we substitute in the reaction above CoA with derivatives containing the acyl group already attached to the phosphopantetheine moiety, then we would be able to produce hexanoyl-ACP (figure on the right) ready to be used directly for the <i> in vitro </i> <del class="diffchange diffchange-inline">the </del>reaction with LuxI. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p> If we substitute in the reaction above CoA with derivatives containing the acyl group already attached to the phosphopantetheine moiety, then we would be able to produce hexanoyl-ACP (figure on the right) ready to be used directly for the <i> in vitro </i> reaction with LuxI. </p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p> There have been some reports on mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6) </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p> There have been some reports on mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6) </p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </p> The behavior of the <del class="diffchange diffchange-inline">enzyme </del>depending on the <del class="diffchange diffchange-inline">substrate </del>is illustrated in figure <del class="diffchange diffchange-inline">3</del>. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </p> The behavior of the <ins class="diffchange diffchange-inline">ACP synthase </ins>depending on the <ins class="diffchange diffchange-inline">CoA and apo-ACP substrates </ins>is illustrated in <ins class="diffchange diffchange-inline">the </ins>figure <ins class="diffchange diffchange-inline">below</ins>. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></p> As a successful example from literature to support our experimental design, the Km value for butyryl production (<del class="diffchange diffchange-inline">fig. 4</del>) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></p> As a successful example from literature to support our experimental design, the Km value for butyryl production (<ins class="diffchange diffchange-inline">figure on the right</ins>) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </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> Detection </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2> Detection </h2></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/2/2a/Gel222222.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/2/2a/Gel222222.jpg" class="border thumb right"></a></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/2/2a/Gel222222.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/2/2a/Gel222222.jpg" class="border thumb right"></a></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p> A first simple step to judge if the reaction is successful would be to run the reaction mixture on a gel. There is evidence that, in spite of a bigger mass, acylated ACP can travel faster than apo or <del class="diffchange diffchange-inline">holoACP </del>in an SDS-PAGE, due to active binding of the SDS detergent molecules to the hydrophobic acyl chain which increases the charge to mass ratio of the entire molecule, thus, facilitating faster migration (<del class="diffchange diffchange-inline">fig. 5</del>) (2,7). On the other hand it is possible to run the reaction mixture also on a native gel and compare the migration pattern of the proteins. The potein that would migrate faster than apoACP in SDS PAGE but slower than it in a native page (separation by mass) would be the acylated ACP <del class="diffchange diffchange-inline">(fig. 6)</del>. Supposing that just a fraction of the ACP interacts to produce the acylated derivative, bands for both hexanoyl-ACP and apo/ holoACP were also expected. Dimerization of holo-ACP would also generate <del class="diffchange diffchange-inline">and additional </del>band) </p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p> A first simple step to judge if the reaction is successful would be to run the reaction mixture on a gel. There is evidence that, in spite of a bigger mass, acylated ACP can travel faster than apo or <ins class="diffchange diffchange-inline">holo ACP </ins>in an SDS-PAGE, due to active binding of the SDS detergent molecules to the hydrophobic acyl chain which increases the charge to mass ratio of the entire molecule, thus, facilitating faster migration (<ins class="diffchange diffchange-inline">figure on the right</ins>) (2,7). On the other hand it is possible to run the reaction mixture also on a native gel and compare the migration pattern of the proteins. The potein that would migrate faster than apoACP in SDS PAGE but slower than it in a native page (separation by mass) would be the acylated ACP. Supposing that just a fraction of the ACP interacts to produce the acylated derivative, bands for both hexanoyl-ACP and apo/ holoACP were also expected. Dimerization of holo-ACP would also generate <ins class="diffchange diffchange-inline">a </ins>band) </p> </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> Although this approach isn’t quantitative, it would give a hint about the efficiency of the reaction. </p> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> Although this approach isn’t quantitative, it would give a hint about the efficiency of the reaction. </p> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p> An intermediary step for the detection of the acyl-ACP synthesis <del class="diffchange diffchange-inline">IS </del>the incubation of the reaction mixture with cell extracts of strains constitutively expressing LuxI. Using as a control LuxI extract incubated with a receiver strain and no reaction mixture, normalization is possible and the relation of fluorescence to <del class="diffchange diffchange-inline">the amount </del>of reaction mix <del class="diffchange diffchange-inline">provided </del>can be tracked. </p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> An intermediary step for the detection of the acyl-ACP synthesis <ins class="diffchange diffchange-inline">is </ins>the incubation of the reaction mixture with cell extracts of strains constitutively expressing LuxI. Using as a control LuxI extract incubated with <ins class="diffchange diffchange-inline">SAM, </ins>a receiver strain and no reaction mixture, normalization is possible and the relation of fluorescence to <ins class="diffchange diffchange-inline">varying amounts </ins>of <ins class="diffchange diffchange-inline">the provided </ins>reaction mix <ins class="diffchange diffchange-inline">containing hexanoyl-ACP </ins>can be tracked. </p> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p> A next integrating approach is to test our system with that of the “Fusion protein” <del class="diffchange diffchange-inline">(yellow team)</del>. If we test our AHL-sensitive reporter cells with SAM, hexanoyl-ACP containing reaction mixture and the recombinant LuxI, then the activation of the reporter cells by this reaction mixture proves the fact that we have produced both functional acyl-ACP and preserved the AHL producing function of the fusion protein. More than this, quantification can be done to assess the maximally efficient conditions for acyl-ACP synthesis </p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> A next integrating approach is to test our system with that of the “Fusion protein”. If we test our AHL-sensitive reporter cells <ins class="diffchange diffchange-inline">again </ins>with SAM, hexanoyl-ACP containing reaction mixture and the recombinant LuxI, then the activation of the reporter cells by this reaction mixture proves the fact that we have produced both functional acyl-ACP and preserved the AHL producing function of the fusion protein. More than this, quantification can be done to assess the maximally efficient conditions for acyl-ACP synthesis </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> Methods </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2> Methods </h2></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p> The reaction components and their molar ratios were adjusted based on previous studies (4,5) and are as follows: 50 mM Tris-HCl, ph 8.0 buffer, 10 mM MgCl2 (enzyme cofactor), 1 mM DTT, 300 µM CoA, 50 µM of apo-ACP and 5 µM of ACP synthase in a total of 100µl reaction volume. The mix is incubated at 37°C for 30 minutes. For terminating the reaction, 50 mM EDTA or 10% trichloroacetic acid is added. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p> The reaction components and their molar ratios were adjusted based on previous studies (4,5) and are as follows: 50 mM Tris-HCl, ph 8.0 buffer, 10 mM MgCl2 (enzyme cofactor), 1 mM DTT, 300 µM CoA, 50 µM of apo-ACP and 5 µM of ACP synthase in a total of 100µl reaction volume. The mix is incubated at 37°C for 30 minutes. For terminating the reaction, 50 mM EDTA or 10% trichloroacetic acid is added. </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> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><h2> References </h2></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">References are listed in the Resource/ Literature section</ins></div></td></tr>
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</table>Vyctoryohttp://2010.igem.org/wiki/index.php?title=Team:BIOTEC_Dresden/ACP&diff=203033&oldid=prevBiba at 00:52, 28 October 20102010-10-28T00:52:02Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2> Detection </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2> Detection </h2></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><ins class="diffchange diffchange-inline"><a href="https://static.igem.org/mediawiki/2010/2/2a/Gel222222.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/2/2a/Gel222222.jpg" class="border thumb right"></a></p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p> A first simple step to judge if the reaction is successful would be to run the reaction mixture on a gel. There is evidence that, in spite of a bigger mass, acylated ACP can travel faster than apo or holoACP in an SDS-PAGE, due to active binding of the SDS detergent molecules to the hydrophobic acyl chain which increases the charge to mass ratio of the entire molecule, thus, facilitating faster migration (fig. 5) (2,7). On the other hand it is possible to run the reaction mixture also on a native gel and compare the migration pattern of the proteins. The potein that would migrate faster than apoACP in SDS PAGE but slower than it in a native page (separation by mass) would be the acylated ACP (fig. 6). Supposing that just a fraction of the ACP interacts to produce the acylated derivative, bands for both hexanoyl-ACP and apo/ holoACP were also expected. Dimerization of holo-ACP would also generate and additional band) </p> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p> A first simple step to judge if the reaction is successful would be to run the reaction mixture on a gel. There is evidence that, in spite of a bigger mass, acylated ACP can travel faster than apo or holoACP in an SDS-PAGE, due to active binding of the SDS detergent molecules to the hydrophobic acyl chain which increases the charge to mass ratio of the entire molecule, thus, facilitating faster migration (fig. 5) (2,7). On the other hand it is possible to run the reaction mixture also on a native gel and compare the migration pattern of the proteins. The potein that would migrate faster than apoACP in SDS PAGE but slower than it in a native page (separation by mass) would be the acylated ACP (fig. 6). Supposing that just a fraction of the ACP interacts to produce the acylated derivative, bands for both hexanoyl-ACP and apo/ holoACP were also expected. Dimerization of holo-ACP would also generate and additional band) </p> </div></td></tr>
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</table>Bibahttp://2010.igem.org/wiki/index.php?title=Team:BIOTEC_Dresden/ACP&diff=202973&oldid=prevBiba at 00:49, 28 October 20102010-10-28T00:49:16Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"> <p> There have been some reports on mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6) </p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" class="border thumb right"></a></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" class="border thumb right"></a></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;"> <p> There have been some reports on mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6) </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 style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p> The behavior of the enzyme depending on the substrate is illustrated in figure 3. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p> The behavior of the enzyme depending on the substrate is illustrated in figure 3. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p></div></td></tr>
</table>Bibahttp://2010.igem.org/wiki/index.php?title=Team:BIOTEC_Dresden/ACP&diff=202942&oldid=prevBiba at 00:47, 28 October 20102010-10-28T00:47:50Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p> There have been some reports on mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6) </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p> There have been some reports on mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6) </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;"><a href="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" class="border thumb right"></a></p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p> The behavior of the enzyme depending on the substrate is illustrated in figure 3. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p> The behavior of the enzyme depending on the substrate is illustrated in figure 3. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><a href="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" class="border thumb right"></a></p></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p> As a successful example from literature to support our experimental design, the Km value for butyryl production (fig. 4) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p> As a successful example from literature to support our experimental design, the Km value for butyryl production (fig. 4) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </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>Bibahttp://2010.igem.org/wiki/index.php?title=Team:BIOTEC_Dresden/ACP&diff=202911&oldid=prevBiba at 00:46, 28 October 20102010-10-28T00:46:57Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p> The behavior of the enzyme depending on the substrate is illustrated in figure 3. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p> The behavior of the enzyme depending on the substrate is illustrated in figure 3. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></p> As a successful example from literature to support our experimental design, the Km value for butyryl production (fig. 4) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" class="border thumb right"></a></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" class="border thumb right"></a></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;"></p> As a successful example from literature to support our experimental design, the Km value for butyryl production (fig. 4) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </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 style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
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</table>Bibahttp://2010.igem.org/wiki/index.php?title=Team:BIOTEC_Dresden/ACP&diff=202876&oldid=prevBiba at 00:45, 28 October 20102010-10-28T00:45:30Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p> As a successful example from literature to support our experimental design, the Km value for butyryl production (fig. 4) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p> As a successful example from literature to support our experimental design, the Km value for butyryl production (fig. 4) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </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;"><a href="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/7/72/Butyryl-CoA.jpg" class="border thumb right"></a></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 style="color: red; font-weight: bold; text-decoration: none;"></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> Detection </h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2> Detection </h2></div></td></tr>
</table>Bibahttp://2010.igem.org/wiki/index.php?title=Team:BIOTEC_Dresden/ACP&diff=202807&oldid=prevBiba at 00:42, 28 October 20102010-10-28T00:42:58Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p> There have been some reports on mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6) </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p> There have been some reports on mild inhibitory activity for this type of reaction by both an excess of apo-ACP and adenosine 3',5'-bisphosphate along with some divalent ions. At the same time Mg2+ or Mn2+ are required for catalytic activity (6) </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p> The behavior of the enzyme depending on the substrate is illustrated in figure 3. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </p> The behavior of the enzyme depending on the substrate is illustrated in figure 3. </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;"><a href="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" rel="lightbox"><img src="https://static.igem.org/mediawiki/2010/9/9f/CoA%2BApo.jpg" class="border thumb left"></a></p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p> As a successful example from literature to support our experimental design, the Km value for butyryl production (fig. 4) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p> As a successful example from literature to support our experimental design, the Km value for butyryl production (fig. 4) from the respective substrates by this strategy was about 8.1 µM which is close to the value of non-derivatized CoA substrate. The turnover number however was 5 times lower than for CoA (0.2 s-1 compared to 1.0 s-1) which could be overcome by longer incubation times. </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>Biba