http://2012.igem.org/wiki/index.php?title=Team:Colombia/Project/Experiments/Ralstonia&feed=atom&action=historyTeam:Colombia/Project/Experiments/Ralstonia - Revision history2024-03-29T00:08:05ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:Colombia/Project/Experiments/Ralstonia&diff=229923&oldid=prevPaolareyescaldas: /* Construction of the PxpsR Reporter */2012-09-27T01:24:58Z<p><span class="autocomment">Construction of the PxpsR Reporter</span></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 01:24, 27 September 2012</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 12:</td>
<td colspan="2" class="diff-lineno">Line 12:</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>==Construction of the PxpsR Reporter==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Construction of the PxpsR Reporter==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In order to quantify the responsiveness of PxpsR to the presence of 3 OH-PAME we generated a PxpsR-eYFP reporter [http://partsregistry.org/Part:BBa_K831017 (K831017)] using the E0430 BioBrick (Image 2). For a more extensive and more detailed explanation of the cloning procedures, please visit: [https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal''<del class="diffchange diffchange-inline">]. The reporter was tested in the pSB1A2 however the part was ensambled into de pSB1C3 backbone and sent to the iGEM Registry Parts. Here we show the basal activity (in absence of 3 OH-PAME) of the PxpsR-eYFP reporter (Image 3) and the [http://partsregistry.org/Part:BBa_K831017 functionality</del>].</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In order to quantify the responsiveness of PxpsR to the presence of 3 OH-PAME we generated a PxpsR-eYFP reporter [http://partsregistry.org/Part:BBa_K831017 (K831017)] using the E0430 BioBrick (Image 2). For a more extensive and more detailed explanation of the cloning procedures, please visit: [https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']. </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>[[File:PxpsR-eYFP.png|250px|thumb|center|''Image 2''. '''Confirmation of PxpsR-eYFP construct'''. After confirmation of colonies by PCR, plasmids of 2 candidates were digest with EcoRI and SpeI. Lane 1, MW (Fermentas 1KB ladder). Lane 2, Candidate 1: promotorless eYFP (E0430). Lane 3, Candidate 2: PxpsR-eYFP.''Note'': We used a reclycled gel to optimize the laboratory reagents.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:PxpsR-eYFP.png|250px|thumb|center|''Image 2''. '''Confirmation of PxpsR-eYFP construct'''. After confirmation of colonies by PCR, plasmids of 2 candidates were digest with EcoRI and SpeI. Lane 1, MW (Fermentas 1KB ladder). Lane 2, Candidate 1: promotorless eYFP (E0430). Lane 3, Candidate 2: PxpsR-eYFP.''Note'': We used a reclycled gel to optimize the laboratory reagents.]]</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">The reporter was tested in the pSB1A2 however the part was ensambled into de pSB1C3 backbone and sent to the iGEM Registry Parts. Here we show the basal activity (in absence of 3 OH-PAME) of the PxpsR-eYFP reporter (Image 3) and the [http://partsregistry.org/Part:BBa_K831017 functionality].</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:PxpsR-YFP.png|450px|thumb|center|''Image 3''. '''Functionality of the reporter PxpsR-eYFP'''. Bacterials strains were photographed while using Differential Interference Contrast (DIC) microscopy and Fluorescence microscopy using FITC filter. Bacteria transformed with PxpsR-eYFP construct show basal levels of expression of the reporter while none of the bacteria transformed with the promotorless eYFP show fluorescence. High background levels in the promotorless eYFP are due to the autoflorescence of the LB medium and the high exposition.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:PxpsR-YFP.png|450px|thumb|center|''Image 3''. '''Functionality of the reporter PxpsR-eYFP'''. Bacterials strains were photographed while using Differential Interference Contrast (DIC) microscopy and Fluorescence microscopy using FITC filter. Bacteria transformed with PxpsR-eYFP construct show basal levels of expression of the reporter while none of the bacteria transformed with the promotorless eYFP show fluorescence. High background levels in the promotorless eYFP are due to the autoflorescence of the LB medium and the high exposition.]]</div></td></tr>
</table>Paolareyescaldashttp://2012.igem.org/wiki/index.php?title=Team:Colombia/Project/Experiments/Ralstonia&diff=224802&oldid=prevPaolareyescaldas: /* Construction of the PxpsR Reporter */2012-09-26T23:45:50Z<p><span class="autocomment">Construction of the PxpsR Reporter</span></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 23:45, 26 September 2012</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 12:</td>
<td colspan="2" class="diff-lineno">Line 12:</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>==Construction of the PxpsR Reporter==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Construction of the PxpsR Reporter==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In order to quantify the responsiveness of PxpsR to the presence of 3 OH-PAME we generated a PxpsR-eYFP reporter<del class="diffchange diffchange-inline">(</del>[http://partsregistry.org/Part:BBa_K831017 (K831017)]<del class="diffchange diffchange-inline">) </del> using the E0430 BioBrick (Image 2). For a more extensive and more detailed explanation of the cloning procedures, please visit: [https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']. The reporter was tested in the pSB1A2 however the part was ensambled into de pSB1C3 backbone and sent to the iGEM Registry Parts. Here we show the <del class="diffchange diffchange-inline">functionality and </del>basal activity (in absence of 3 OH-PAME) of the PxpsR-eYFP reporter (Image 3).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In order to quantify the responsiveness of PxpsR to the presence of 3 OH-PAME we generated a PxpsR-eYFP reporter [http://partsregistry.org/Part:BBa_K831017 (K831017)] using the E0430 BioBrick (Image 2). For a more extensive and more detailed explanation of the cloning procedures, please visit: [https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']. The reporter was tested in the pSB1A2 however the part was ensambled into de pSB1C3 backbone and sent to the iGEM Registry Parts. Here we show the basal activity (in absence of 3 OH-PAME) of the PxpsR-eYFP reporter (Image 3) <ins class="diffchange diffchange-inline">and the [http://partsregistry.org/Part:BBa_K831017 functionality]</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:PxpsR-eYFP.png|250px|thumb|center|''Image 2''. '''Confirmation of PxpsR-eYFP construct'''. After confirmation of colonies by PCR, plasmids of 2 candidates were digest with EcoRI and SpeI. Lane 1, MW (Fermentas 1KB ladder). Lane 2, Candidate 1: promotorless eYFP (E0430). Lane 3, Candidate 2: PxpsR-eYFP.''Note'': We used a reclycled gel to optimize the laboratory reagents.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:PxpsR-eYFP.png|250px|thumb|center|''Image 2''. '''Confirmation of PxpsR-eYFP construct'''. After confirmation of colonies by PCR, plasmids of 2 candidates were digest with EcoRI and SpeI. Lane 1, MW (Fermentas 1KB ladder). Lane 2, Candidate 1: promotorless eYFP (E0430). Lane 3, Candidate 2: PxpsR-eYFP.''Note'': We used a reclycled gel to optimize the laboratory reagents.]]</div></td></tr>
</table>Paolareyescaldashttp://2012.igem.org/wiki/index.php?title=Team:Colombia/Project/Experiments/Ralstonia&diff=224636&oldid=prevPaolareyescaldas: /* PxpsR Reporter */2012-09-26T23:42:40Z<p><span class="autocomment">PxpsR Reporter</span></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 23:42, 26 September 2012</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 11:</td>
<td colspan="2" class="diff-lineno">Line 11:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:ConfpxpsrdigxbaIspeI.png|250px|thumb|center|''Image 1''. '''Confirmation of PxpsR BioBrick'''. Lane 1, MW (Fermentas 1KB ladder). Lane 2, PxpsR cloned in pBS1C3 digest with XbaI and SpeI. Lane 3, Positive control of PxpsR (PCR product).''Note'': We used a reclycled gel to optimize the laboratory reagents.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:ConfpxpsrdigxbaIspeI.png|250px|thumb|center|''Image 1''. '''Confirmation of PxpsR BioBrick'''. Lane 1, MW (Fermentas 1KB ladder). Lane 2, PxpsR cloned in pBS1C3 digest with XbaI and SpeI. Lane 3, Positive control of PxpsR (PCR product).''Note'': We used a reclycled gel to optimize the laboratory reagents.]]</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>==PxpsR Reporter==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>==<ins class="diffchange diffchange-inline">Construction of the </ins>PxpsR Reporter==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In order to quantify the responsiveness of PxpsR to the presence of 3 OH-PAME we generated a PxpsR-eYFP reporter([http://partsregistry.org/Part:BBa_K831017 (K831017)]) using the E0430 BioBrick (Image 2). For a more extensive and more detailed explanation of the cloning procedures, please visit: [https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']. The reporter was tested in the pSB1A2 however the part was ensambled into de pSB1C3 backbone and sent to the iGEM Registry Parts. Here we show the functionality and basal activity (in absence of 3 OH-PAME) of the PxpsR-eYFP reporter (Image 3).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In order to quantify the responsiveness of PxpsR to the presence of 3 OH-PAME we generated a PxpsR-eYFP reporter([http://partsregistry.org/Part:BBa_K831017 (K831017)]) using the E0430 BioBrick (Image 2). For a more extensive and more detailed explanation of the cloning procedures, please visit: [https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']. The reporter was tested in the pSB1A2 however the part was ensambled into de pSB1C3 backbone and sent to the iGEM Registry Parts. Here we show the functionality and basal activity (in absence of 3 OH-PAME) of the PxpsR-eYFP reporter (Image 3).</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>Paolareyescaldashttp://2012.igem.org/wiki/index.php?title=Team:Colombia/Project/Experiments/Ralstonia&diff=224570&oldid=prevPaolareyescaldas: /* Ralstonia BioBricks */2012-09-26T23:41:35Z<p><span class="autocomment">Ralstonia BioBricks</span></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 23:41, 26 September 2012</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 5:</td>
<td colspan="2" class="diff-lineno">Line 5:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=''Ralstonia'' Experiments=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=''Ralstonia'' Experiments=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==''Ralstonia'' BioBricks==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==''Ralstonia'' BioBricks==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We cloned and generated a biobrick([http://partsregistry.org/Part:BBa_K831016 K831016]) for the promoter region of xpsR (PxpsR). The product of this gene is an integrator signal that regulates the expression of several virulence factors in ''Ralstonia solanacearum'' (Shell, 1996). One of the signals that XpsR integrates is phcA which is involved in the Quorum Sensing mediated by 3 OH-PAME exclusive of ''R. solanacearum''. We designed primers to amplify the upstream region of xpsR gene including the two binding boxes of phcA, the -35 and -10 conserved boxes and excluding the native RBS (Figure 1). This part was confirmed by sequencing. In addition, we cloned the three genes (phcA, phcS and phcR) involved in the sensing of the 3 OH-PAME (<del class="diffchange diffchange-inline">Gerin </del>and Denny, 2012), further confirmation of these parts is still needed. Image 1 shows the enzymatic confirmation for PxpsR in the backbone pBS1C3. For a more extensive and more detailed description of the cloning procedures, please visit: [https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We cloned and generated a biobrick([http://partsregistry.org/Part:BBa_K831016 K831016]) for the promoter region of xpsR (PxpsR). The product of this gene is an integrator signal that regulates the expression of several virulence factors in ''Ralstonia solanacearum'' (Shell, 1996). One of the signals that XpsR integrates is phcA which is involved in the Quorum Sensing mediated by 3 OH-PAME exclusive of ''R. solanacearum''. We designed primers to amplify the upstream region of xpsR gene including the two binding boxes of phcA, the -35 and -10 conserved boxes and excluding the native RBS (Figure 1). This part was confirmed by sequencing. In addition, we cloned the three genes (phcA, phcS and phcR) involved in the sensing of the 3 OH-PAME (<ins class="diffchange diffchange-inline">Genin </ins>and Denny, 2012), further confirmation of these parts is still needed. Image 1 shows the enzymatic confirmation for PxpsR in the backbone pBS1C3. For a more extensive and more detailed description of the cloning procedures, please visit: [https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']</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>[[File:Captura_de_pantalla_2012-09-26_a_las_16.55.14.png|300px|thumb|center|''Figure 1.''''' xpsR Promoter Region''' Modified from Huang ''et al.'', 1998. Figure shows the boxes upstream in the promoter region of xpsR gene.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Captura_de_pantalla_2012-09-26_a_las_16.55.14.png|300px|thumb|center|''Figure 1.''''' xpsR Promoter Region''' Modified from Huang ''et al.'', 1998. Figure shows the boxes upstream in the promoter region of xpsR gene.]]</div></td></tr>
</table>Paolareyescaldashttp://2012.igem.org/wiki/index.php?title=Team:Colombia/Project/Experiments/Ralstonia&diff=224551&oldid=prevPaolareyescaldas: /* Ralstonia BioBricks */2012-09-26T23:41:10Z<p><span class="autocomment">Ralstonia BioBricks</span></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 23:41, 26 September 2012</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 5:</td>
<td colspan="2" class="diff-lineno">Line 5:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=''Ralstonia'' Experiments=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=''Ralstonia'' Experiments=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==''Ralstonia'' BioBricks==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==''Ralstonia'' BioBricks==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We cloned and generated a biobrick([http://partsregistry.org/Part:BBa_K831016 K831016]) for the promoter region of xpsR (PxpsR). The product of this gene is an integrator signal that regulates the expression of several virulence factors in ''Ralstonia solanacearum''(Shell, 1996). One of the signals that XpsR integrates is phcA which is involved in the Quorum Sensing mediated by 3 OH-PAME exclusive of ''R. solanacearum''. We designed primers to amplify the upstream region of xpsR gene including the two binding boxes of phcA, the -35 and -10 conserved boxes and excluding the native RBS (Figure 1). This part was confirmed by sequencing. In addition, we cloned the three genes (phcA, phcS and phcR) involved in the sensing of the 3 OH-PAME(Gerin and <del class="diffchange diffchange-inline">Shell</del>, 2012), further confirmation of these parts is still needed. Image 1 shows the enzymatic confirmation for PxpsR in the backbone pBS1C3. For a more extensive and more detailed description of the cloning procedures, please visit: [https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We cloned and generated a biobrick([http://partsregistry.org/Part:BBa_K831016 K831016]) for the promoter region of xpsR (PxpsR). The product of this gene is an integrator signal that regulates the expression of several virulence factors in ''Ralstonia solanacearum'' (Shell, 1996). One of the signals that XpsR integrates is phcA which is involved in the Quorum Sensing mediated by 3 OH-PAME exclusive of ''R. solanacearum''. We designed primers to amplify the upstream region of xpsR gene including the two binding boxes of phcA, the -35 and -10 conserved boxes and excluding the native RBS (Figure 1). This part was confirmed by sequencing. In addition, we cloned the three genes (phcA, phcS and phcR) involved in the sensing of the 3 OH-PAME (Gerin and <ins class="diffchange diffchange-inline">Denny</ins>, 2012), further confirmation of these parts is still needed. Image 1 shows the enzymatic confirmation for PxpsR in the backbone pBS1C3. For a more extensive and more detailed description of the cloning procedures, please visit: [https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']</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>[[File:Captura_de_pantalla_2012-09-26_a_las_16.55.14.png|300px|thumb|center|''Figure 1.''''' xpsR Promoter Region''' Modified from Huang ''et al.'', 1998. Figure shows the boxes upstream in the promoter region of xpsR gene.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Captura_de_pantalla_2012-09-26_a_las_16.55.14.png|300px|thumb|center|''Figure 1.''''' xpsR Promoter Region''' Modified from Huang ''et al.'', 1998. Figure shows the boxes upstream in the promoter region of xpsR gene.]]</div></td></tr>
</table>Paolareyescaldashttp://2012.igem.org/wiki/index.php?title=Team:Colombia/Project/Experiments/Ralstonia&diff=224507&oldid=prevPaolareyescaldas: /* Ralstonia BioBricks */2012-09-26T23:40:17Z<p><span class="autocomment">Ralstonia BioBricks</span></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 23:40, 26 September 2012</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 5:</td>
<td colspan="2" class="diff-lineno">Line 5:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=''Ralstonia'' Experiments=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=''Ralstonia'' Experiments=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==''Ralstonia'' BioBricks==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==''Ralstonia'' BioBricks==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We cloned and generated a biobrick([http://partsregistry.org/Part:BBa_K831016 K831016]) for the promoter region of xpsR (PxpsR). The product of this gene is an integrator signal that regulates the expression of several virulence factors in ''Ralstonia solanacearum''(). One of the signals that XpsR integrates is phcA which is involved in the Quorum Sensing mediated by 3 OH-PAME exclusive of ''R. solanacearum''. We designed primers to amplify the upstream region of xpsR gene including the two binding boxes of phcA, the -35 and -10 conserved boxes and excluding the native RBS (Figure 1). This part was confirmed by sequencing. In addition, we cloned the three genes (phcA, phcS and phcR) involved in the sensing of the 3 OH-PAME(), further confirmation of these parts is still needed. Image 1 shows the enzymatic confirmation for PxpsR in the backbone pBS1C3. For a more extensive and more detailed description of the cloning procedures, please visit: [https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We cloned and generated a biobrick([http://partsregistry.org/Part:BBa_K831016 K831016]) for the promoter region of xpsR (PxpsR). The product of this gene is an integrator signal that regulates the expression of several virulence factors in ''Ralstonia solanacearum''(<ins class="diffchange diffchange-inline">Shell, 1996</ins>). One of the signals that XpsR integrates is phcA which is involved in the Quorum Sensing mediated by 3 OH-PAME exclusive of ''R. solanacearum''. We designed primers to amplify the upstream region of xpsR gene including the two binding boxes of phcA, the -35 and -10 conserved boxes and excluding the native RBS (Figure 1). This part was confirmed by sequencing. In addition, we cloned the three genes (phcA, phcS and phcR) involved in the sensing of the 3 OH-PAME(<ins class="diffchange diffchange-inline">Gerin and Shell, 2012</ins>), further confirmation of these parts is still needed. Image 1 shows the enzymatic confirmation for PxpsR in the backbone pBS1C3. For a more extensive and more detailed description of the cloning procedures, please visit: [https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']</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>[[File:Captura_de_pantalla_2012-09-26_a_las_16.55.14.png|300px|thumb|center|''Figure 1.''''' xpsR Promoter Region''' Modified from Huang ''et al.'', 1998. Figure shows the boxes upstream in the promoter region of xpsR gene.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Captura_de_pantalla_2012-09-26_a_las_16.55.14.png|300px|thumb|center|''Figure 1.''''' xpsR Promoter Region''' Modified from Huang ''et al.'', 1998. Figure shows the boxes upstream in the promoter region of xpsR gene.]]</div></td></tr>
</table>Paolareyescaldashttp://2012.igem.org/wiki/index.php?title=Team:Colombia/Project/Experiments/Ralstonia&diff=224464&oldid=prevPaolareyescaldas: /* References */2012-09-26T23:39:30Z<p><span class="autocomment">References</span></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 23:39, 26 September 2012</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 36:</td>
<td colspan="2" class="diff-lineno">Line 36:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=References=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=References=</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>#Schell, M. A. (1996) To be or not to be: how Pseudomonas solanacearum decides whether or not to express virulence genes. Eur. J. Plant Pathol. 102:459–469.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Schell, M. A. (1996) To be or not to be: how<ins class="diffchange diffchange-inline">'' </ins>Pseudomonas solanacearum<ins class="diffchange diffchange-inline">'' </ins>decides whether or not to express virulence genes. Eur. J. Plant Pathol. 102:459–469.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Huang J., Yindeeyoungyeon W., Garg R.P., Denny T.P. and Schell M.A. (1998) Joint Transcriptional Control of xpsR, the Unusual Signal Integrator of the Ralstonia solanacearum Virulence Gene Regulatory Network, by a Response Regulator and a LysR-Type Transcriptional Activator. J. Bacteriol. 180(10): 2736-2743</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#Genin S. and Denny T.P. (2012) Pathogenomics of the ''Ralstonia solanacearum'' Species Complex. Ann. Rev. Phytopath., 50: 67 -89</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#Huang J., Yindeeyoungyeon W., Garg R.P., Denny T.P. and Schell M.A. (1998) Joint Transcriptional Control of xpsR, the Unusual Signal Integrator of the <ins class="diffchange diffchange-inline">''</ins>Ralstonia solanacearum<ins class="diffchange diffchange-inline">'' </ins>Virulence Gene Regulatory Network, by a Response Regulator and a LysR-Type Transcriptional Activator. J. Bacteriol. 180(10): 2736-2743</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></div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></td></tr>
</table>Paolareyescaldashttp://2012.igem.org/wiki/index.php?title=Team:Colombia/Project/Experiments/Ralstonia&diff=224108&oldid=prevPaolareyescaldas: /* References */2012-09-26T23:33:09Z<p><span class="autocomment">References</span></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 23:33, 26 September 2012</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 36:</td>
<td colspan="2" class="diff-lineno">Line 36:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=References=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=References=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#<del class="diffchange diffchange-inline">Stefan D. Muri</del>, <del class="diffchange diffchange-inline">Hilko van der Voet</del>, <del class="diffchange diffchange-inline">Polly E</del>. <del class="diffchange diffchange-inline">Boon</del>, <del class="diffchange diffchange-inline">Jacob D</del>. <del class="diffchange diffchange-inline">van Klaveren</del>, <del class="diffchange diffchange-inline">Beat J</del>. <del class="diffchange diffchange-inline">Brüschweiler</del>. <del class="diffchange diffchange-inline">Comparison of human healthrisks resulting from exposure to fungicides </del>and <del class="diffchange diffchange-inline">mycotoxins via food Food and Chemical Toxicology</del>, <del class="diffchange diffchange-inline">Volume 47, Issue 12, December 2009</del>, <del class="diffchange diffchange-inline">Pages 2963–2974</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#<ins class="diffchange diffchange-inline">Schell</ins>, <ins class="diffchange diffchange-inline">M. A. (1996) To be or not to be: how Pseudomonas solanacearum decides whether or not to express virulence genes. Eur. J. Plant Pathol. 102:459–469.</ins></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">#Huang J.</ins>, <ins class="diffchange diffchange-inline">Yindeeyoungyeon W</ins>., <ins class="diffchange diffchange-inline">Garg R.P</ins>., <ins class="diffchange diffchange-inline">Denny T</ins>.<ins class="diffchange diffchange-inline">P</ins>. and <ins class="diffchange diffchange-inline">Schell M.A. (1998) Joint Transcriptional Control of xpsR</ins>, <ins class="diffchange diffchange-inline">the Unusual Signal Integrator of the Ralstonia solanacearum Virulence Gene Regulatory Network</ins>, <ins class="diffchange diffchange-inline">by a Response Regulator and a LysR-Type Transcriptional Activator. J. Bacteriol. 180(10): 2736-2743</ins></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>
</table>Paolareyescaldashttp://2012.igem.org/wiki/index.php?title=Team:Colombia/Project/Experiments/Ralstonia&diff=223773&oldid=prevPaolareyescaldas: /* Ralstonia Experiments */2012-09-26T23:26:37Z<p><span class="autocomment">Ralstonia Experiments</span></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 23:26, 26 September 2012</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 33:</td>
<td colspan="2" class="diff-lineno">Line 33:</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>[[File:MethodsRalstonia.png|700px|thumb|center|''Figure 4''. '''Experiment carried out to determine the cuantitative induction of the promoter PxpsR'''.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:MethodsRalstonia.png|700px|thumb|center|''Figure 4''. '''Experiment carried out to determine the cuantitative induction of the promoter PxpsR'''.]]</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">=References=</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">#Stefan D. Muri, Hilko van der Voet, Polly E. Boon, Jacob D. van Klaveren, Beat J. Brüschweiler. Comparison of human healthrisks resulting from exposure to fungicides and mycotoxins via food Food and Chemical Toxicology, Volume 47, Issue 12, December 2009, Pages 2963–2974</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;"></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>Paolareyescaldashttp://2012.igem.org/wiki/index.php?title=Team:Colombia/Project/Experiments/Ralstonia&diff=221123&oldid=prevPaolareyescaldas: /* PxpsR Reporter */2012-09-26T22:31:59Z<p><span class="autocomment">PxpsR Reporter</span></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 22:31, 26 September 2012</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 12:</td>
<td colspan="2" class="diff-lineno">Line 12:</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>==PxpsR Reporter==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==PxpsR Reporter==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In order to quantify the responsiveness of PxpsR to the presence of 3 OH-PAME we generated a PxpsR-eYFP reporter using the E0430 BioBrick (Image 2) For a more extensive and more detailed cloning procedures <del class="diffchange diffchange-inline">here </del>[https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']. The reporter was tested in the pSB1A2 however the part was ensambled into de pSB1C3 backbone and sent to the iGEM Registry Parts. Here we show the functionality and basal activity (in absence of 3 OH-PAME) of the PxpsR-eYFP reporter (Image 3).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In order to quantify the responsiveness of PxpsR to the presence of 3 OH-PAME we generated a PxpsR-eYFP reporter<ins class="diffchange diffchange-inline">([http://partsregistry.org/Part:BBa_K831017 (K831017)]) </ins>using the E0430 BioBrick (Image 2)<ins class="diffchange diffchange-inline">. </ins>For a more extensive and more detailed <ins class="diffchange diffchange-inline">explanation of the </ins>cloning procedures<ins class="diffchange diffchange-inline">, please visit: </ins>[https://2012.igem.org/Team:Colombia/Notebook/Journal ''Ralstonia Journal'']. The reporter was tested in the pSB1A2 however the part was ensambled into de pSB1C3 backbone and sent to the iGEM Registry Parts. Here we show the functionality and basal activity (in absence of 3 OH-PAME) of the PxpsR-eYFP reporter (Image 3).</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>[[File:PxpsR-eYFP.png|250px|thumb|center|''Image 2''. '''Confirmation of PxpsR-eYFP construct'''. After confirmation of colonies by PCR, plasmids of 2 candidates were digest with EcoRI and SpeI. Lane 1, MW (Fermentas 1KB ladder). Lane 2, Candidate 1: promotorless eYFP (E0430). Lane 3, Candidate 2: PxpsR-eYFP.''Note'': We used a reclycled gel to optimize the laboratory reagents.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:PxpsR-eYFP.png|250px|thumb|center|''Image 2''. '''Confirmation of PxpsR-eYFP construct'''. After confirmation of colonies by PCR, plasmids of 2 candidates were digest with EcoRI and SpeI. Lane 1, MW (Fermentas 1KB ladder). Lane 2, Candidate 1: promotorless eYFP (E0430). Lane 3, Candidate 2: PxpsR-eYFP.''Note'': We used a reclycled gel to optimize the laboratory reagents.]]</div></td></tr>
</table>Paolareyescaldas