http://2012.igem.org/wiki/index.php?title=Team:Valencia/genetic_engineering&feed=atom&action=historyTeam:Valencia/genetic engineering - Revision history2024-03-28T22:29:14ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:Valencia/genetic_engineering&diff=238093&oldid=prevDaie07 at 03:52, 27 September 20122012-09-27T03:52:23Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure 4). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure 4). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Figure 4: </b> pAM977 transformants</center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Figure 4: </b> pAM977 transformants</center></div></td></tr>
</table>Daie07http://2012.igem.org/wiki/index.php?title=Team:Valencia/genetic_engineering&diff=237149&oldid=prevAurfor at 03:38, 27 September 20122012-09-27T03:38:21Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195, table 1) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195, table 1) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <ins class="diffchange diffchange-inline"><br></ins><br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Table 1: </b> Information of the two vectors used for characterize the <i>psbAI</i> promoter</center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Table 1: </b> Information of the two vectors used for characterize the <i>psbAI</i> promoter</center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br></div></td></tr>
</table>Aurforhttp://2012.igem.org/wiki/index.php?title=Team:Valencia/genetic_engineering&diff=236950&oldid=prevRoocfer at 03:35, 27 September 20122012-09-27T03:35:31Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> (figure 1) for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> (figure 1) for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div align="right"><b>Figure 1: </b> <i>S. elongatus</i> PCC7942 </div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div align="right"><b>Figure 1: </b> <i>S. elongatus</i> PCC7942 </div></div></td></tr>
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</table>Roocferhttp://2012.igem.org/wiki/index.php?title=Team:Valencia/genetic_engineering&diff=236711&oldid=prevRoocfer at 03:31, 27 September 20122012-09-27T03:31:05Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b>Cloning into <i>E. coli</i></b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b>Cloning into <i>E. coli</i></b<ins class="diffchange diffchange-inline">></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><center><b>Figure 2: </b>pAM2195 vector</center><br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><center><b>Figure 2: </b>pAM2195 vector</center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b>Transforming Coccus</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We used the following protocol to transform <i>Synechococcus</i>: <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We used the following protocol to transform <i>Synechococcus</i>: <br><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Protocol</b></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>Protocol</b></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Extracted from Clerico et al. 2007:<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Extracted from Clerico et al. 2007:<br></div></td></tr>
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</table>Roocferhttp://2012.igem.org/wiki/index.php?title=Team:Valencia/genetic_engineering&diff=236580&oldid=prevRoocfer at 03:28, 27 September 20122012-09-27T03:28:58Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b>Cloning into <i>E. coli</i></b></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b>Cloning into <i>E. coli</i></b<ins class="diffchange diffchange-inline">><br</ins>></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Figure 2: </b>pAM2195 vector</center><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Figure 2: </b>pAM2195 vector</center><br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b>Transforming Coccus</b></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b>Transforming Coccus</b<ins class="diffchange diffchange-inline">><br</ins>></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br></div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 52:</td>
<td colspan="2" class="diff-lineno">Line 52:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Figure 3: </b>A view of our transformed cells</center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Figure 3: </b>A view of our transformed cells</center></div></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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><h2></del><b>Results</b<del class="diffchange diffchange-inline">></h2</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b>Results</b></div></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;"><div>We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Figure 4: </b> pAM977 transformants</center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Figure 4: </b> pAM977 transformants</center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><br></ins><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></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><h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br></div></td></tr>
</table>Roocferhttp://2012.igem.org/wiki/index.php?title=Team:Valencia/genetic_engineering&diff=236450&oldid=prevRoocfer at 03:26, 27 September 20122012-09-27T03:26:52Z<p></p>
<table style="background-color: white; color:black;">
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<col class='diff-marker' />
<col class='diff-content' />
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 03:26, 27 September 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><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 class="diffchange diffchange-inline"><h2></del><b>Cloning into <i>E. coli</i></b<del class="diffchange diffchange-inline">></h2</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b>Cloning into <i>E. coli</i></b></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Figure 2: </b>pAM2195 vector</center><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Figure 2: </b>pAM2195 vector</center><br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><h2></del><b>Transforming Coccus</b<del class="diffchange diffchange-inline">></h2</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b>Transforming Coccus</b></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><h2></del><b>Protocol</b<del class="diffchange diffchange-inline">></h2</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b>Protocol</b></div></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;"><div>Extracted from Clerico et al. 2007:<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Extracted from Clerico et al. 2007:<br></div></td></tr>
</table>Roocferhttp://2012.igem.org/wiki/index.php?title=Team:Valencia/genetic_engineering&diff=236361&oldid=prevRoocfer at 03:25, 27 September 20122012-09-27T03:25:32Z<p></p>
<table style="background-color: white; color:black;">
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 03:25, 27 September 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Cloning into <i>E. coli</i></b></h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Cloning into <i>E. coli</i></b></h2></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/<del class="diffchange diffchange-inline">LB_Agar</del>">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/<ins class="diffchange diffchange-inline">Transforming_ecoli</ins>">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Figure 2: </b>pAM2195 vector</center><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><b>Figure 2: </b>pAM2195 vector</center><br><br></div></td></tr>
</table>Roocferhttp://2012.igem.org/wiki/index.php?title=Team:Valencia/genetic_engineering&diff=236329&oldid=prevAurfor at 03:24, 27 September 20122012-09-27T03:24:57Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Cloning into <i>E. coli</i></b></h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Cloning into <i>E. coli</i></b></h2></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/LB_Agar">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/LB_Agar">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> </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"><center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br></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"><center><b>Figure 2: </b>pAM2195 vector</center><br><br></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Transforming Coccus</b></h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Transforming Coccus</b></h2></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. </div></td></tr>
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<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;"><div>We used the following protocol to transform <i>Synechococcus</i>: <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We used the following protocol to transform <i>Synechococcus</i>: <br><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 class="diffchange diffchange-inline"><center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br></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"><center><b>Figure 2: </b>pAM2195 vector</center></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><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;"><div><h2><b>Protocol</b></h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Protocol</b></h2></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Atsumi, S., Higashide, W., and Liao, J. C. (2009) Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. <i>Nat Biotechnol</i>. 27:1177-1180<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Atsumi, S., Higashide, W., and Liao, J. C. (2009) Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. <i>Nat Biotechnol</i>. 27:1177-1180<br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Clerico, E. M., Ditty, J. L. & Golden, S.S. (2007) Specialized Techniques for Site-Directed Mutagenesis in Cyanobacteria. <i>Methods in Molecular Biology.</i> 362:153–172.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Clerico, E. M., Ditty, J. L. & Golden, S.S. (2007) Specialized Techniques for Site-Directed Mutagenesis in Cyanobacteria. <i>Methods in Molecular Biology.</i> 362:153–172.<br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Wang, B., Wang J., Zhang, W. & Meldrum, D. R. (2012) Application of Synthetic Biology in cyanobacteria and algae. Frontiers in Microbiology<del class="diffchange diffchange-inline">. </del>doi: 10.3389/fmicb.2012.00344<br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Nair, U., Thomas, C. & Golden, S. S. (2001) Functional Elements of the Strong <i>psbAI</i> Promoter of Synechococcus elongatus PCC 7942. <i>J. Bacteriology</i>, 183:1740–1747.<br><br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Wang, B., Wang J., Zhang, W. & Meldrum, D. R. (2012) Application of Synthetic Biology in cyanobacteria and algae. Frontiers in Microbiology<ins class="diffchange diffchange-inline">, </ins>doi: 10.3389/fmicb.2012.00344<br><br></div></td></tr>
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</table>Aurforhttp://2012.igem.org/wiki/index.php?title=Team:Valencia/genetic_engineering&diff=235818&oldid=prevRoocfer at 03:16, 27 September 20122012-09-27T03:16:58Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Cloning into <i>E. coli</i></b></h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Cloning into <i>E. coli</i></b></h2></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this protocol <del class="diffchange diffchange-inline">(link)</del>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <ins class="diffchange diffchange-inline"><a href="https://2012.igem.org/Team:Valencia/LB_Agar"></ins>protocol<ins class="diffchange diffchange-inline"></a></ins>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Transforming Coccus</b></h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Transforming Coccus</b></h2></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Extracted from Clerico et al. 2007:<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Extracted from Clerico et al. 2007:<br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li>We grew 100ml of cscB and WT in BG-11 liquid medium <del class="diffchange diffchange-inline">(see how we grown them and our recipe here) </del>shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li>We grew 100ml of cscB and WT in BG-11 liquid medium shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a JetQuick kit <del class="diffchange diffchange-inline">(link al kit?)</del></li><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a <ins class="diffchange diffchange-inline"><i></ins>JetQuick<ins class="diffchange diffchange-inline"></i> </ins>kit </li><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar <del class="diffchange diffchange-inline">(see here the recipe) </del>with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions.<a href="https://2012.igem.org/Team:Valencia/prototypes"> Here </a> you will find all the information concerning the different parts of the BioBrick.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions.<a href="https://2012.igem.org/Team:Valencia/prototypes"> Here </a> you will find all the information concerning the different parts of the BioBrick.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We successfully cloned all the parts in DH5α E. coli strains using this protocol <del class="diffchange diffchange-inline">(link)</del>. We used the BioBrick Assembly Manual <del class="diffchange diffchange-inline">(link) </del>protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We successfully cloned all the parts in DH5α E. coli strains using this <ins class="diffchange diffchange-inline"><a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli"></ins>protocol<ins class="diffchange diffchange-inline"></a></ins>. We used the <ins class="diffchange diffchange-inline"><a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf"></ins>BioBrick Assembly Manual<ins class="diffchange diffchange-inline"></a> </ins>protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br></div></td></tr>
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</table>Roocferhttp://2012.igem.org/wiki/index.php?title=Team:Valencia/genetic_engineering&diff=235362&oldid=prevAurfor at 03:08, 27 September 20122012-09-27T03:08:55Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br><br><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br><br><br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><div align="right"><b>Figure 1:</b> <i>S. elongatus</i> PCC7942 </div></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><div align="right"><b>Figure 1: </b> <i>S. elongatus</i> PCC7942 </div></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195, table 1) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195, table 1) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><center><b>Table 1:</b> Information of the two vectors used for characterize the <i>psbAI</i> promoter</center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><center><b>Table 1: </b> Information of the two vectors used for characterize the <i>psbAI</i> promoter</center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><center><b>Figure 2:</b>pAM2195 vector</center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><center><b>Figure 2: </b>pAM2195 vector</center></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Protocol</b></h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Protocol</b></h2></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><center><b>Figure 3:</b>A view of our transformed cells</center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><center><b>Figure 3: </b>A view of our transformed cells</center></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Results</b></h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2><b>Results</b></h2></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><center><b>Figure 4:</b> pAM977 transformants</center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><center><b>Figure 4: </b> pAM977 transformants</center></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><center><b>Table 2:</b> <del class="diffchange diffchange-inline">Description</del></center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><center><b>Table 2: </b> <ins class="diffchange diffchange-inline">Experimental design for AHL production </ins></center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><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><br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab <del class="diffchange diffchange-inline">WAS NOT VIBRIO FISCHERI</del>, but <del class="diffchange diffchange-inline">VIBRIO MEDITERRANEI </del>(GREAT!).<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab <ins class="diffchange diffchange-inline">was not <i> V. fischeri</i></ins>, but <ins class="diffchange diffchange-inline"><i>V.mediterrani</i> </ins>(GREAT!).<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We will try to obtain <i>V. fischeri</i> as soon as possible to test this<del class="diffchange diffchange-inline">.</del>.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We will try to obtain <i>V. fischeri</i> as soon as possible to test this.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2012/6/6b/Electro_VLCXXX.jpg" width="350" height="270"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><img src="https://static.igem.org/mediawiki/2012/6/6b/Electro_VLCXXX.jpg" width="350" height="270"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><center><b>Figures 6 & 7:</b> Succesfully we build our BioBrick. In the electrophoresis the band that corresponds to the part (3868bp) is the one surrounded with a red triangle </center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><center><b>Figures 6 & 7: </b> Succesfully we build our BioBrick. In the electrophoresis the band that corresponds to the part (3868bp) is the one surrounded with a red triangle </center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><center><b>Figure 8:</b> <del class="diffchange diffchange-inline">Description</del></center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><center><b>Figure 8: </b> <ins class="diffchange diffchange-inline"><i>psbAI</i> promoter inside the pSB1C3</ins></center></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><strong>References</strong></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><strong>References</strong></div></td></tr>
</table>Aurfor