http://2009.igem.org/wiki/index.php?title=Team:Groningen/Project/Transport&feed=atom&action=historyTeam:Groningen/Project/Transport - Revision history2024-03-29T11:19:35ZRevision history for this page on the wikiMediaWiki 1.16.5http://2009.igem.org/wiki/index.php?title=Team:Groningen/Project/Transport&diff=159231&oldid=prevNienke: /* Results */2009-10-21T23:16:56Z<p><span class="autocomment">Results</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 23:16, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 43:</td>
<td colspan="2" class="diff-lineno">Line 43:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The graphs above represent the result of the metal sensitivity [https://2009.igem.org/Team:Groningen/Protocols#Death_assay assay]. The lines in the graphs represent the average optical density (OD<sub>600</sub>) of a construct over time. The graph on the left shows that increased As(III) levels inhibits growth and as the As(III) concentration increases the plateau OD<sub>600</sub> level decreases. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The graphs above represent the result of the metal sensitivity [https://2009.igem.org/Team:Groningen/Protocols#Death_assay assay]. The lines in the graphs represent the average optical density (OD<sub>600</sub>) of a construct over time. The graph on the left shows that increased As(III) levels inhibits growth and as the As(III) concentration increases the plateau OD<sub>600</sub> level decreases. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The second graph represents the OD<sub>600</sub> values of a ''E. coli'' culture with pLac-GlpF. The <del class="diffchange diffchange-inline">growht </del>curves are less steep in the log phase compared to WT, because of the protein expression by IPTG induction. In the absence of As(III) the plateau level equals the WT, but if arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) which implies functional expression of GlpF causing As(III) uptake to toxic levels. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The second graph represents the OD<sub>600</sub> values of a ''E. coli'' culture with pLac-GlpF. The <ins class="diffchange diffchange-inline">growth </ins>curves are less steep in the log phase compared to WT, because of the protein expression by IPTG induction. In the absence of As(III) the plateau level equals the WT, but if arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) which implies functional expression of GlpF causing As(III) uptake to toxic levels. </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>In the graph on the right, shows growth curves of ''E. coli'' expressing GlpF and fMT under a low constitutive promoter. Also a similar growth in the log phase, compared to pLac GlpF, can be seen this is due to protein expression. Furthermore, like WT (0μM As(III)) it has its plateau above OD<sub>600</sub> 0.9. Upon incubation with various arsenite concentrations, the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. The reduced growth is also an indicator for arsenite uptake. It is difficult to see if fMT has any effect because this assay can not show where the arsenite is and how fMT interferes with the cells' detoxification.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the graph on the right, shows growth curves of ''E. coli'' expressing GlpF and fMT under a low constitutive promoter. Also a similar growth in the log phase, compared to pLac GlpF, can be seen this is due to protein expression. Furthermore, like WT (0μM As(III)) it has its plateau above OD<sub>600</sub> 0.9. Upon incubation with various arsenite concentrations, the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. The reduced growth is also an indicator for arsenite uptake. It is difficult to see if fMT has any effect because this assay can not show where the arsenite is and how fMT interferes with the cells' detoxification.</div></td></tr>
</table>Nienkehttp://2009.igem.org/wiki/index.php?title=Team:Groningen/Project/Transport&diff=159197&oldid=prevNienke: /* Results */2009-10-21T23:15:47Z<p><span class="autocomment">Results</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 23:15, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 41:</td>
<td colspan="2" class="diff-lineno">Line 41:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:DeathAssayLow.png|310px|left]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:DeathAssayLow.png|310px|left]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The graphs above represent the result of the metal sensitivity [https://2009.igem.org/Team:Groningen/Protocols#Death_assay assay]. The lines in the graphs represent the average optical density of a construct over time. The graph on the left <del class="diffchange diffchange-inline">show </del>that increased As(III) levels <del class="diffchange diffchange-inline">inhibit </del>growth and<del class="diffchange diffchange-inline">, that </del>as <del class="diffchange diffchange-inline">more </del>As(III) <del class="diffchange diffchange-inline">is added the lower </del>the plateau <del class="diffchange diffchange-inline">is</del>. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The graphs above represent the result of the metal sensitivity [https://2009.igem.org/Team:Groningen/Protocols#Death_assay assay]. The lines in the graphs represent the average optical density <ins class="diffchange diffchange-inline">(OD<sub>600</sub>) </ins>of a construct over time. The graph on the left <ins class="diffchange diffchange-inline">shows </ins>that increased As(III) levels <ins class="diffchange diffchange-inline">inhibits </ins>growth and as <ins class="diffchange diffchange-inline">the </ins>As(III) <ins class="diffchange diffchange-inline">concentration increases </ins>the plateau <ins class="diffchange diffchange-inline">OD<sub>600</sub> level decreases</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: #ffa; color:black; font-size: smaller;"><div>The <del class="diffchange diffchange-inline">middle </del>graph <del class="diffchange diffchange-inline">is from </del>the pLac GlpF <del class="diffchange diffchange-inline">construct</del>. The curves are less steep in the log phase compared to WT because of the protein <del class="diffchange diffchange-inline">expresion </del>by IPTG induction. In the absence of As(III) the plateau level equals the WT<del class="diffchange diffchange-inline">. If </del>arsenite is present the plateaus are lower <del class="diffchange diffchange-inline"> </del>(OD<sub>600</sub> <0.8) <del class="diffchange diffchange-inline">compared to WT. This is due to </del>As(III) uptake <del class="diffchange diffchange-inline">by GlpF</del>. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The <ins class="diffchange diffchange-inline">second </ins>graph <ins class="diffchange diffchange-inline">represents </ins>the <ins class="diffchange diffchange-inline">OD<sub>600</sub> values of a ''E. coli'' culture with </ins>pLac<ins class="diffchange diffchange-inline">-</ins>GlpF. The <ins class="diffchange diffchange-inline">growht </ins>curves are less steep in the log phase compared to WT<ins class="diffchange diffchange-inline">, </ins>because of the protein <ins class="diffchange diffchange-inline">expression </ins>by IPTG induction. In the absence of As(III) the plateau level equals the WT<ins class="diffchange diffchange-inline">, but if </ins>arsenite is present the plateaus are lower (OD<sub>600</sub> <0.8) <ins class="diffchange diffchange-inline">which implies functional expression of GlpF causing </ins>As(III) uptake <ins class="diffchange diffchange-inline">to toxic levels</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: #ffa; color:black; font-size: smaller;"><div>In the graph on the right <del class="diffchange diffchange-inline">we see the </del>curves of <del class="diffchange diffchange-inline">low constitutively expressed </del>GlpF and fMT <del class="diffchange diffchange-inline">and it shows </del>a similar <del class="diffchange diffchange-inline">slope </del>in the log phase compared to pLac GlpF due to protein expression <del class="diffchange diffchange-inline">and </del>like WT <del class="diffchange diffchange-inline">0 μM </del>As(III) it has its plateau <del class="diffchange diffchange-inline">over </del>OD<sub>600</sub> 0.9. <del class="diffchange diffchange-inline">If </del>arsenite <del class="diffchange diffchange-inline">is present </del>the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. <del class="diffchange diffchange-inline">Here the </del>reduced growth is also an indicator for arsenite uptake. It is difficult to see if fMT has <del class="diffchange diffchange-inline">an </del>effect because this assay can not show where the arsenite is and how fMT interferes with the cells <del class="diffchange diffchange-inline">detoxificatoin</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In the graph on the right<ins class="diffchange diffchange-inline">, shows growth </ins>curves of <ins class="diffchange diffchange-inline">''E. coli'' expressing </ins>GlpF and fMT <ins class="diffchange diffchange-inline">under a low constitutive promoter. Also </ins>a similar <ins class="diffchange diffchange-inline">growth </ins>in the log phase<ins class="diffchange diffchange-inline">, </ins>compared to pLac GlpF<ins class="diffchange diffchange-inline">, can be seen this is </ins>due to protein expression<ins class="diffchange diffchange-inline">. Furthermore, </ins>like WT <ins class="diffchange diffchange-inline">(0μM </ins>As(III<ins class="diffchange diffchange-inline">)</ins>) it has its plateau <ins class="diffchange diffchange-inline">above </ins>OD<sub>600</sub> 0.9. <ins class="diffchange diffchange-inline">Upon incubation with various </ins>arsenite <ins class="diffchange diffchange-inline">concentrations, </ins>the plateaus are lower (OD<sub>600</sub> <0.8) compared to WT. This is due to As(III) uptake by GlpF. <ins class="diffchange diffchange-inline">The </ins>reduced growth is also an indicator for arsenite uptake. It is difficult to see if fMT has <ins class="diffchange diffchange-inline">any </ins>effect because this assay can not show where the arsenite is and how fMT interferes with the cells<ins class="diffchange diffchange-inline">' detoxification</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>==={{anchor|Modelling}}Modelling uptake GlpF===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==={{anchor|Modelling}}Modelling uptake GlpF===</div></td></tr>
</table>Nienkehttp://2009.igem.org/wiki/index.php?title=Team:Groningen/Project/Transport&diff=158055&oldid=prevJelle: /* Additional sources */2009-10-21T22:44:25Z<p><span class="autocomment">Additional sources</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 22:44, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 149:</td>
<td colspan="2" class="diff-lineno">Line 149:</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;"></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>===Additional sources===</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">=</ins>===Additional sources<ins class="diffchange diffchange-inline">=</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>More information on this topic can be found in:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>More information on this topic can be found in:</div></td></tr>
</table>Jellehttp://2009.igem.org/wiki/index.php?title=Team:Groningen/Project/Transport&diff=157796&oldid=prevJelle: /* Results */2009-10-21T22:36:19Z<p><span class="autocomment">Results</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 22:36, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 139:</td>
<td colspan="2" class="diff-lineno">Line 139:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>====Results==== </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>====Results==== </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HmtA_SDS_gel.jpg|200px|thumb|right|[Team:Groningen/Team|HmtA-6HIS on SDS-page]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HmtA_SDS_gel.jpg|200px|thumb|right|[Team:Groningen/Team|HmtA-6HIS on SDS-page]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>So far we have cloned HmtA as a biobrick without EcoRI site in the coding region into the iGEM vector. Unfortunately a mutation occurred at base 103 from the start of the orf. By a point mutation c to t in the first nucleotide of the codon changed arginine 35 to a Cysteine. We do not know the effects but we suspect it might have a great influence due to the very reactive side chain of Cysteine, eventhough it is not in the channel itself based on [http://www.cbs.dtu.dk/services/TMHMM/ TMHMM] predictions which indicate trans membrane helices of a protein. Further cloning is expected to be unsuccessful because the <del class="diffchange diffchange-inline">iPTG </del>induced clones grow even slightly better than the empty vector control. This is most likely cause by the missing pLAC-RBS in front of the gene. There was no positive controle with the L-arabinose inducable HmtA-6His in pBAD. We did do expression experiments with the pBAD construct to purified the membrane protein as quality controle. result shown in the figure on the right.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>So far we have cloned HmtA as a biobrick without EcoRI site in the coding region into the iGEM vector. Unfortunately a mutation occurred at base 103 from the start of the orf. By a point mutation c to t in the first nucleotide of the codon changed arginine 35 to a Cysteine. We do not know the effects but we suspect it might have a great influence due to the very reactive side chain of Cysteine, eventhough it is not in the channel itself based on [http://www.cbs.dtu.dk/services/TMHMM/ TMHMM] predictions which indicate trans membrane helices of a protein. Further cloning is expected to be unsuccessful because the <ins class="diffchange diffchange-inline">IPTG </ins>induced clones grow even slightly better than the empty vector control. This is most likely cause by the missing pLAC-RBS in front of the gene. There was no positive controle with the L-arabinose inducable HmtA-6His in pBAD. We did do expression experiments with the pBAD construct to purified the membrane protein as quality controle. result shown in the figure on the right.</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>==Heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM==</div></td></tr>
</table>Jellehttp://2009.igem.org/wiki/index.php?title=Team:Groningen/Project/Transport&diff=156429&oldid=prevFrans: /* Export of arsenicum via Ars operon */2009-10-21T21:48:32Z<p><span class="autocomment">Export of arsenicum via Ars operon</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 21:48, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 112:</td>
<td colspan="2" class="diff-lineno">Line 112:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></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>==Export of arsenicum via Ars operon==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">=</ins>==Export of arsenicum via Ars operon<ins class="diffchange diffchange-inline">=</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>GlpF is the importer of arsenicum. After arsenicum enters the cell, in response the Ars operon produces ArsR. At the same time, ArsB is also produced by Ars operon. This happens because the Ars operon contains three open reading frames: the first is ArsR, second ArsB and the last one is ArsC. ArsB is the exporter of arsenicum. The ars operon is located on the chromosomal DNA of E. coli.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>GlpF is the importer of arsenicum. After arsenicum enters the cell, in response the Ars operon produces ArsR. At the same time, ArsB is also produced by Ars operon. This happens because the Ars operon contains three open reading frames: the first is ArsR, second ArsB and the last one is ArsC. ArsB is the exporter of arsenicum. The ars operon is located on the chromosomal DNA of E. coli.</div></td></tr>
</table>Franshttp://2009.igem.org/wiki/index.php?title=Team:Groningen/Project/Transport&diff=156401&oldid=prevFrans: /* {{anchor|Modelling}}Modelling uptake GlpF */2009-10-21T21:47:36Z<p><span class="autocomment">{{anchor|Modelling}}Modelling uptake GlpF</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 21:47, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 112:</td>
<td colspan="2" class="diff-lineno">Line 112:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></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"> </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;">==Export of arsenicum via Ars operon==</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 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;">GlpF is the importer of arsenicum. After arsenicum enters the cell, in response the Ars operon produces ArsR. At the same time, ArsB is also produced by Ars operon. This happens because the Ars operon contains three open reading frames: the first is ArsR, second ArsB and the last one is ArsC. ArsB is the exporter of arsenicum. The ars operon is located on the chromosomal DNA of E. coli.</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;">For more information see: [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE-IN-CHROM-BROWSER&object=EG12235 biocyc].</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;">[[Image:ArsRBC_operon.PNG|600px]]</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>===Additional sources===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Additional sources===</div></td></tr>
</table>Franshttp://2009.igem.org/wiki/index.php?title=Team:Groningen/Project/Transport&diff=156367&oldid=prevFrans: /* Export of arsenicum via Ars operon */2009-10-21T21:46:38Z<p><span class="autocomment">Export of arsenicum via Ars operon</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 21:46, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 158:</td>
<td colspan="2" class="diff-lineno">Line 158:</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>As the cells die after uptake of Mg (and induction of the Mer transporter), this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem ([[Team:Groningen/Literature#Pennella2005|Pennella 2005]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As the cells die after uptake of Mg (and induction of the Mer transporter), this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem ([[Team:Groningen/Literature#Pennella2005|Pennella 2005]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<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;">==Export of arsenicum via Ars operon==</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">GlpF is the importer of arsenicum. After arsenicum enters the cell, in response the Ars operon produces ArsR. At the same time, ArsB is also produced by Ars operon. This happens because the Ars operon contains three open reading frames: the first is ArsR, second ArsB and the last one is ArsC. ArsB is the exporter of arsenicum. The ars operon is located on the chromosomal DNA of E. coli.</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;">For more information see: [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE-IN-CHROM-BROWSER&object=EG12235 biocyc].</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[[Image:ArsRBC_operon.PNG|600px]]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #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;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>Franshttp://2009.igem.org/wiki/index.php?title=Team:Groningen/Project/Transport&diff=156309&oldid=prevFrans: /* Planning and requirements */2009-10-21T21:44:53Z<p><span class="autocomment">Planning and requirements</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 21:44, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 161:</td>
<td colspan="2" class="diff-lineno">Line 161:</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;"></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;">==Planning and requirements==</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">* '''Modelling:'''</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;">** Import speed</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;">** Amount </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;">** Max</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;">* '''Lab:'''</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;">** HmtA</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;">*** Zn/Cu alone</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;">*** B-type ATPase (could be use if there is a ATP shortage?)</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;">** CitM (probably not used)</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;">*** Divalent ions</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;">*** Citrate around</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;">*** Citrate can bind metals that are already bound.</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;">** Measurements (both for the "normal" cell and the cell with overexpression of the transporter)</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;">*** Transporter, on/off mechanism, up to what concentration (in the cell) does it still have metal uptake.</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;">*** Measure concentration of metal. difference between begin and end concentrations of metal outside the cell.</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;">*** How fast does it transport metal in/out the cell.</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;">**** Set up tests with (initial) extracellular concentrations of about <sup>1</sup>/<sub>3</sub>K (25% of V<sub>max</sub>), K (50% of V<sub>max</sub>), 3K (75% of V<sub>max</sub>) and 10mM (99.7% of V<sub>max</sub>, corresponding to extremely polluted water), and a control with no arsenic. Obviously, more tests is better. In general a desired fraction of V<sub>max</sub> at the initial concentration can be attained by using an initial concentration of x/(1-x) times K.</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;">**** Determine "final" (steady-state) concentration of As(III) in the solution and in the cells. (Concentration over time is even better!)</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;">**** This means that the total volume of the cells (and the solution) has to be determined. Possibly through looking at the dry weight (without arsenic!).</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;">**** By manipulating the equation for the derivative of As(III) in equilibrium, As(III) can be expressed as a function of As(III)<sub>ex</sub> (given the V and K constants). We can try to fill in the computed V and K constants for GlpF and then use a least squares fit to estimate the V and K constants for ArsB.</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;">**** '''NOTE:''' Interestingly [[Team:Groningen/Literature#Kostal2004|Kostal 2004]] already did an experiment like this with cells that overexpressed ArsR. We're looking at analysing these results under the assumption that overexpressing ArsR only gives a constant factor more accumulation (for 1-100&microM As(III)), but it would be very nice to do this ourselves for unmodified cells to determine whether this is indeed true (and to determine the factor).</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Export of arsenicum via Ars operon==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Export of arsenicum via Ars operon==</div></td></tr>
</table>Franshttp://2009.igem.org/wiki/index.php?title=Team:Groningen/Project/Transport&diff=156091&oldid=prevFrans: /* Missing information/To Do */2009-10-21T21:38:51Z<p><span class="autocomment">Missing information/To Do</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 21:38, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 159:</td>
<td colspan="2" class="diff-lineno">Line 159:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As the cells die after uptake of Mg (and induction of the Mer transporter), this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem ([[Team:Groningen/Literature#Pennella2005|Pennella 2005]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As the cells die after uptake of Mg (and induction of the Mer transporter), this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem ([[Team:Groningen/Literature#Pennella2005|Pennella 2005]], [[Team:Groningen/Literature#Kao2008|Kao 2008]]).</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">===Missing information/To Do===</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">*Expression assesment</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">**Stability</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">**Level</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">*Functional assesment</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">**Uptake speed</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">**Affinity</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">**Electrolyte potential generating force</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">*Eliminate BioBrick restriction sites</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;"></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>==Planning and requirements==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Planning and requirements==</div></td></tr>
</table>Franshttp://2009.igem.org/wiki/index.php?title=Team:Groningen/Project/Transport&diff=155795&oldid=prevJelle: /* Missing information/To Do */2009-10-21T21:28:38Z<p><span class="autocomment">Missing information/To Do</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 21:28, 21 October 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 112:</td>
<td colspan="2" class="diff-lineno">Line 112:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">===Missing information/To Do===</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;">*Expression assesment</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;">**Stability</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;">**Level</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;">*Functional assesment</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;">**Uptake speed</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;">**Affinity</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;">**Electrolyte potential generating force</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">*<del>Q:Eliminate BioBrick restriction sites</del></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">*<del>Q: What does the ars operon of our <i>E. coli</i> look like? Do we have both ArsA and ArsB? (And what about ArsR and ArsD?)</del> A: We only have ArsRBC, see [[Team:Groningen/BLAST|our BLAST results]].</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><br></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Additional sources===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Additional sources===</div></td></tr>
</table>Jelle