http://2011.igem.org/wiki/index.php?title=Team:Brown-Stanford/PowerCell/NutrientSecretion&feed=atom&action=historyTeam:Brown-Stanford/PowerCell/NutrientSecretion - Revision history2024-03-28T17:58:17ZRevision history for this page on the wikiMediaWiki 1.16.0http://2011.igem.org/wiki/index.php?title=Team:Brown-Stanford/PowerCell/NutrientSecretion&diff=262984&oldid=prevEvanclark: /* PowerCell Transformation */2011-12-07T09:55:32Z<p><span class="autocomment">PowerCell Transformation</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>After about 2 weeks of growth, we can confirm the correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of the GFP reporter. GFP expression is on in vegetative cells, and off in heterocysts, which implies that the sugar secretion construct is only active in vegetative cells, as we desire. Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations will be high enough to support growth of E. coli W; our experiments suggest the minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids<del class="diffchange diffchange-inline">, </del>to show that PowerCell can power general biological tools. Updates will be posted here as they become available! </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>After about 2 weeks of growth, we can confirm the correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of the GFP reporter. GFP expression is on in vegetative cells, and off in heterocysts, which implies that the sugar secretion construct is only active in vegetative cells, as we desire. Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations will be high enough to support growth of E. coli W; our experiments suggest the minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids to show that PowerCell can power general biological tools. Updates will be posted here as they become available! </div></td></tr>
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</table>Evanclarkhttp://2011.igem.org/wiki/index.php?title=Team:Brown-Stanford/PowerCell/NutrientSecretion&diff=262983&oldid=prevEvanclark: /* PowerCell Transformation */2011-12-07T09:54:33Z<p><span class="autocomment">PowerCell Transformation</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Anabaena Selection.jpeg|400px||center|thumb|Left: BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Middle: BG11(N-) with neomycin + 50µl of the original conjugation mixture. Right: BG11(N-) with neomycin + 50µl of WT Anabaena.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Anabaena Selection.jpeg|400px||center|thumb|Left: BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Middle: BG11(N-) with neomycin + 50µl of the original conjugation mixture. Right: BG11(N-) with neomycin + 50µl of WT Anabaena.</div></td></tr>
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<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 initial selection looked promising. The left-most culture tube contains BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Both transformed and untransformed Anabaena should be able to grow in this mixture. As expected, this culture tube has the highest cell density. The middle culture tube contains BG11(N-) with neomycin + 50µl of the original conjugation mixture. Only Anabaena containing our plasmid should be able to grow in this mixture. As desired, growth is still visible, but is less dense. The right-most tube contains BG11(N-) with neomycin + 50µl of WT Anabaena. Nothing should be able to grow here. As expected, everything in this tube is dead.</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">The initial selection looked promising. The left-most culture tube contains BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Both transformed and untransformed </del>Anabaena <del class="diffchange diffchange-inline">should be able to grow in this mixture. As expected, this culture tube has the highest cell density. The middle culture tube contains BG11(N-) </del>with <del class="diffchange diffchange-inline">neomycin + 50µl of the original conjugation mixture</del>. <del class="diffchange diffchange-inline">Only Anabaena containing our plasmid should be able </del>to <del class="diffchange diffchange-inline">grow in this mixture. As desired, growth is still visible, but is less dense. The right-most tube contains BG11(N-) with neomycin + 50µl of WT Anabaena. Nothing should be able to grow here. As expected, everything in this tube is dead.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><div style="width: 35%;"></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">Transformed </ins>Anabaena with <ins class="diffchange diffchange-inline">heterocysts stained by alcian blue</ins>. <ins class="diffchange diffchange-inline">Heterocyst spacing corresponds </ins>to the <ins class="diffchange diffchange-inline">"off" cells from </ins>the GFP <ins class="diffchange diffchange-inline">image</ins>, <ins class="diffchange diffchange-inline">implying </ins>that the <ins class="diffchange diffchange-inline">sucrose </ins>secretion construct <ins class="diffchange diffchange-inline">in not </ins>active in <ins class="diffchange diffchange-inline">heterocysts</ins>, as <ins class="diffchange diffchange-inline">desired</ins>.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">After about 2 weeks of growth, we can confirm </del>the <del class="diffchange diffchange-inline">correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of </del>the GFP <del class="diffchange diffchange-inline">reporter. GFP expression is on in vegetative cells, and off in heterocysts</del>, <del class="diffchange diffchange-inline">which implies </del>that the <del class="diffchange diffchange-inline">sugar </del>secretion construct <del class="diffchange diffchange-inline">is only </del>active in <del class="diffchange diffchange-inline">vegetative cells</del>, as <del class="diffchange diffchange-inline">we desire</del>. <del class="diffchange diffchange-inline">Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations will be high enough to support growth of E. coli W; our experiments suggest the minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids, to show that PowerCell can power general biological tools. Updates will be posted here as they become available! </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"></div></ins></div></td></tr>
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<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;">After about 2 weeks of growth, we can confirm the correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of the GFP reporter. GFP expression is on in vegetative cells, and off in heterocysts, which implies that the sugar secretion construct is only active in vegetative cells, as we desire. Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations will be high enough to support growth of E. coli W; our experiments suggest the minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids, to show that PowerCell can power general biological tools. Updates will be posted here as they become available! </ins></div></td></tr>
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</table>Evanclarkhttp://2011.igem.org/wiki/index.php?title=Team:Brown-Stanford/PowerCell/NutrientSecretion&diff=262982&oldid=prevEvanclark: /* PowerCell Transformation */2011-12-07T09:49:44Z<p><span class="autocomment">PowerCell Transformation</span></p>
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</table>Evanclarkhttp://2011.igem.org/wiki/index.php?title=Team:Brown-Stanford/PowerCell/NutrientSecretion&diff=262981&oldid=prevEvanclark: /* PowerCell Transformation */2011-12-07T09:48:29Z<p><span class="autocomment">PowerCell Transformation</span></p>
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<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;">Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.</ins></div></td></tr>
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</table>Evanclarkhttp://2011.igem.org/wiki/index.php?title=Team:Brown-Stanford/PowerCell/NutrientSecretion&diff=262980&oldid=prevEvanclark: /* PowerCell Transformation */2011-12-07T09:47:02Z<p><span class="autocomment">PowerCell Transformation</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After about 2 weeks of growth, we can confirm the correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of the GFP reporter. GFP expression is on in vegetative cells, and off in heterocysts, which implies that the sugar secretion construct is only active in vegetative cells, as we desire. Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations will be high enough to support growth of E. coli W; our experiments suggest the minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids, to show that PowerCell can power general biological tools. Updates will be posted here as they become available! </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After about 2 weeks of growth, we can confirm the correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of the GFP reporter. GFP expression is on in vegetative cells, and off in heterocysts, which implies that the sugar secretion construct is only active in vegetative cells, as we desire. Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations will be high enough to support growth of E. coli W; our experiments suggest the minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids, to show that PowerCell can power general biological tools. Updates will be posted here as they become available! </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td></tr>
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</table>Evanclarkhttp://2011.igem.org/wiki/index.php?title=Team:Brown-Stanford/PowerCell/NutrientSecretion&diff=262979&oldid=prevEvanclark: /* PowerCell Transformation */2011-12-07T09:43:57Z<p><span class="autocomment">PowerCell Transformation</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Brown-Stanford Transformed Anabaena fluorescent.JPG|400px||center|thumb|Same Anabaena culture showing GFP expression in successful transformant]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Brown-Stanford Transformed Anabaena fluorescent.JPG|400px||center|thumb|Same Anabaena culture showing GFP expression in successful transformant]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful<ins class="diffchange diffchange-inline">. Some non-transformants can be seen in the background</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><del class="diffchange diffchange-inline">Correct regulation by the psaC promoter appears to be occurring, as several evenly spaced non-fluorescing cells can be seen along the filament. This is expected due to the presence of heterocysts. However, The image shows the transformation mixture directly after conjugation but before section, so we cannot conclusively state that the non-fluorescing cells are heterocysts as opposed to merely non-</del>transformants<del class="diffchange diffchange-inline">. Yet the results are very promising. Selection is currently underway</del>, and <del class="diffchange diffchange-inline">we will definitively verify correct regulation by the psaC promoter as well as function of the cscB gene very soon.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">We selected for our </ins>transformants <ins class="diffchange diffchange-inline">using neomycin</ins>, and <ins class="diffchange diffchange-inline">let </ins>the <ins class="diffchange diffchange-inline">cultures grow</ins>.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Below is an image of </del>the <del class="diffchange diffchange-inline">selection underway</del>. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Anabaena Selection.jpeg|400px||center|thumb|Left: BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Middle: BG11(N-) with neomycin + 50µl of the original conjugation mixture. Right: BG11(N-) with neomycin + 50µl of WT Anabaena.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Anabaena Selection.jpeg|400px||center|thumb|Left: BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Middle: BG11(N-) with neomycin + 50µl of the original conjugation mixture. Right: BG11(N-) with neomycin + 50µl of WT Anabaena.</div></td></tr>
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<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;">The initial selection looks promising. The left-most culture tube contains BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Both transformed and untransformed Anabaena should be able to grow in this mixture. As expected, this culture tube has the highest cell density. The middle culture tube contains BG11(N-) with neomycin + 50µl of the original conjugation mixture. Only Anabaena containing our plasmid should be able to grow in this mixture. As desired, growth is still visible, but is less dense. The right-most tube contains BG11(N-) with neomycin + 50µl of WT Anabaena. Nothing should be able to grow here. As expected, everything in this tube is dead.</del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Updates on </del>the <del class="diffchange diffchange-inline">further characterization </del>of the <del class="diffchange diffchange-inline">psaC promoter</del>, <del class="diffchange diffchange-inline">cscB gene</del>, and construct as <del class="diffchange diffchange-inline">a whole </del>will be <del class="diffchange diffchange-inline">posted </del>to <del class="diffchange diffchange-inline">this page and </del>the <del class="diffchange diffchange-inline">registry </del>as they become available.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">The initial selection looked promising. The left-most culture tube contains BG11(N-) w/o neomycin selection + 50µl of </ins>the <ins class="diffchange diffchange-inline">original conjugation mixture. Both transformed and untransformed Anabaena should be able to grow in this mixture. As expected, this culture tube has the highest cell density. The middle culture tube contains BG11(N-) with neomycin + 50µl </ins>of the <ins class="diffchange diffchange-inline">original conjugation mixture. Only Anabaena containing our plasmid should be able to grow in this mixture. As desired</ins>, <ins class="diffchange diffchange-inline">growth is still visible, but is less dense. The right-most tube contains BG11(N-) with neomycin + 50µl of WT Anabaena. Nothing should be able to grow here. As expected, everything in this tube is dead.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">After about 2 weeks of growth, we can confirm the correct regulation of our sugar secretion construct by our pSac promoter by observing the presence of the GFP reporter. GFP expression is on in vegetative cells</ins>, and <ins class="diffchange diffchange-inline">off in heterocysts, which implies that the sugar secretion </ins>construct <ins class="diffchange diffchange-inline">is only active in vegetative cells, </ins>as <ins class="diffchange diffchange-inline">we desire. Work is currently underway to assay the sucrose concentrations that are generated by our construct. We hope that these concentrations </ins>will be <ins class="diffchange diffchange-inline">high enough </ins>to <ins class="diffchange diffchange-inline">support growth of E. coli W; our experiments suggest </ins>the <ins class="diffchange diffchange-inline">minimum concentration is around 5 mM (see section below). If the concentrations are high enough, we intend to grow E. coli W hosting several arbitrary active BioBrick plasmids, to show that PowerCell can power general biological tools. Updates will be posted here </ins>as they become available<ins class="diffchange diffchange-inline">! </ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><a href="http://farm8</ins>.<ins class="diffchange diffchange-inline">staticflickr.com/7030/6467315137_890d8a769b_o.jpg"><img src="http://farm8.staticflickr.com/7030/6467315137_890d8a769b_o.jpg" style="width:40%"></ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Transformed Anabaena showing cell-type specific GFP fluorescence under the control of the pSac promoter. GFP expression is on in vegetative cells, off in heterocysts. Some background fluorescence from chlorophyll is visible.</ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><a href="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg"><img src="http://farm8.staticflickr.com/7152/6467315073_227d36b07a_o.jpg" style="width:40%"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"></a></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><br></ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Transformed Anabaena with heterocysts stained with alcian blue</ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"></html></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>Evanclarkhttp://2011.igem.org/wiki/index.php?title=Team:Brown-Stanford/PowerCell/NutrientSecretion&diff=260574&oldid=prevEvanclark: /* PowerCell Transformation */2011-10-29T02:23:28Z<p><span class="autocomment">PowerCell Transformation</span></p>
<table style="background-color: white; color:black;">
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<td colspan='2' style="background-color: white; color:black;">Revision as of 02:23, 29 October 2011</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful.</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>Correct regulation by the psaC promoter appears to be occurring, as several evenly spaced non-fluorescing cells can be seen along the filament. This is expected due to the presence of heterocysts. However, The image shows the transformation mixture directly after conjugation but before section, so we cannot conclusively state that the non-fluorescing cells are heterocysts as opposed to merely non-transformants. Yet the results are very promising. Selection is currently underway, and we will <del class="diffchange diffchange-inline">be able to </del>definitively verify <del class="diffchange diffchange-inline">function of the cscB gene as well as </del>correct regulation by the psaC promoter very soon.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Correct regulation by the psaC promoter appears to be occurring, as several evenly spaced non-fluorescing cells can be seen along the filament. This is expected due to the presence of heterocysts. However, The image shows the transformation mixture directly after conjugation but before section, so we cannot conclusively state that the non-fluorescing cells are heterocysts as opposed to merely non-transformants. Yet the results are very promising. Selection is currently underway, and we will definitively verify correct regulation by the psaC promoter <ins class="diffchange diffchange-inline">as well as function of the cscB gene </ins>very soon.</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>Below is an image of the selection underway. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Below is an image of the selection underway. </div></td></tr>
</table>Evanclarkhttp://2011.igem.org/wiki/index.php?title=Team:Brown-Stanford/PowerCell/NutrientSecretion&diff=260552&oldid=prevEvanclark: /* PowerCell Transformation */2011-10-29T02:22:30Z<p><span class="autocomment">PowerCell Transformation</span></p>
<table style="background-color: white; color:black;">
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful.</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>Correct regulation by the psaC promoter appears to be occurring, as several evenly spaced non-fluorescing cells can be seen along the filament. This <del class="diffchange diffchange-inline">would be </del>expected due to the presence of heterocysts. However, The image shows the transformation mixture directly after conjugation but before section, so we cannot conclusively state that the non-fluorescing cells are heterocysts as opposed to merely non-transformants. Yet the results are very promising. Selection is currently underway, and we will be able to definitively verify function of the cscB gene as well as correct regulation by the psaC promoter very soon.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Correct regulation by the psaC promoter appears to be occurring, as several evenly spaced non-fluorescing cells can be seen along the filament. This <ins class="diffchange diffchange-inline">is </ins>expected due to the presence of heterocysts. However, The image shows the transformation mixture directly after conjugation but before section, so we cannot conclusively state that the non-fluorescing cells are heterocysts as opposed to merely non-transformants. Yet the results are very promising. Selection is currently underway, and we will be able to definitively verify function of the cscB gene as well as correct regulation by the psaC promoter very soon.</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>Below is an image of the selection underway. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Below is an image of the selection underway. </div></td></tr>
</table>Evanclarkhttp://2011.igem.org/wiki/index.php?title=Team:Brown-Stanford/PowerCell/NutrientSecretion&diff=260542&oldid=prevEvanclark: /* PowerCell Transformation */2011-10-29T02:21:46Z<p><span class="autocomment">PowerCell Transformation</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Brown-Stanford Transformed Anabaena fluorescent.JPG|400px||center|thumb|Same Anabaena culture showing GFP expression in successful transformant]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Brown-Stanford Transformed Anabaena fluorescent.JPG|400px||center|thumb|Same Anabaena culture showing GFP expression in successful transformant]]</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;">We transformed the final PowerCell construct into Anabeana using the methods described above. Expression of GFP is clearly visible, indicating the transformation was successful.</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;">Correct regulation by the psaC promoter appears to be occurring, as several evenly spaced non-fluorescing cells can be seen along the filament. This would be expected due to the presence of heterocysts. However, The image shows the transformation mixture directly after conjugation but before section, so we cannot conclusively state that the non-fluorescing cells are heterocysts as opposed to merely non-transformants. Yet the results are very promising. Selection is currently underway, and we will be able to definitively verify function of the cscB gene as well as correct regulation by the psaC promoter very soon.</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;">Below is an image of the selection underway. </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;">[[File:Anabaena Selection.jpeg|400px||center|thumb|Left: BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Middle: BG11(N-) with neomycin + 50µl of the original conjugation mixture. Right: BG11(N-) with neomycin + 50µl of WT Anabaena.</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;"></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 initial selection looks promising. The left-most culture tube contains BG11(N-) w/o neomycin selection + 50µl of the original conjugation mixture. Both transformed and untransformed Anabaena should be able to grow in this mixture. As expected, this culture tube has the highest cell density. The middle culture tube contains BG11(N-) with neomycin + 50µl of the original conjugation mixture. Only Anabaena containing our plasmid should be able to grow in this mixture. As desired, growth is still visible, but is less dense. The right-most tube contains BG11(N-) with neomycin + 50µl of WT Anabaena. Nothing should be able to grow here. As expected, everything in this tube is dead.</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;">Updates on the further characterization of the psaC promoter, cscB gene, and construct as a whole will be posted to this page and the registry as they become available.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>----</div></td></tr>
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</table>Evanclarkhttp://2011.igem.org/wiki/index.php?title=Team:Brown-Stanford/PowerCell/NutrientSecretion&diff=259830&oldid=prevEvanclark: /* Utilizing sucrose */2011-10-29T01:39:13Z<p><span class="autocomment">Utilizing sucrose</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>== '''Utilizing sucrose''' ==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>== '''Utilizing sucrose <ins class="diffchange diffchange-inline">from PowerCell</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>==='''E. coli W''' ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==='''E. coli W''' ===</div></td></tr>
</table>Evanclark