http://2014.igem.org/wiki/index.php?title=Team:SDU-Denmark/Tour40&feed=atom&action=historyTeam:SDU-Denmark/Tour40 - Revision history2024-03-28T11:40:27ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:SDU-Denmark/Tour40&diff=399871&oldid=prevUlrikasimone at 03:46, 18 October 20142014-10-18T03:46:02Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h4>Controlling the Tet promoter</h4></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h4>Controlling the Tet promoter</h4></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Figure <del class="diffchange diffchange-inline">4</del>: Plate containing 200 ng/mL doxycycline plated with <i>E. coli</i> WT and <i>E. coli</i> expressing GFP controlled by a constitutive promoter, pTet-TeR(+LVA) and pTet-TetR(no LVA).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Figure <ins class="diffchange diffchange-inline">5</ins>: Plate containing 200 ng/mL doxycycline plated with <i>E. coli</i> WT and <i>E. coli</i> expressing GFP controlled by a constitutive promoter, pTet-TeR(+LVA) and pTet-TetR(no LVA).</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Figure <del class="diffchange diffchange-inline">5</del>: Plate containing 0 ng/mL doxycycline plated with <i>E. coli</i> WT and <i>E. coli</i> expressing GFP controlled by a constitutive promoter, pTet-TeR(+LVA) and pTet-TetR(no LVA).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Figure <ins class="diffchange diffchange-inline">4</ins>: Plate containing 0 ng/mL doxycycline plated with <i>E. coli</i> WT and <i>E. coli</i> expressing GFP controlled by a constitutive promoter, pTet-TeR(+LVA) and pTet-TetR(no LVA).</div></td></tr>
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</table>Ulrikasimonehttp://2014.igem.org/wiki/index.php?title=Team:SDU-Denmark/Tour40&diff=399780&oldid=prevUlrikasimone at 03:45, 18 October 20142014-10-18T03:45:20Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Figure <del class="diffchange diffchange-inline">2</del>: Plate containing 200 ng/mL doxycycline plated with <i>E. coli</i> WT and <i>E. coli</i> expressing GFP controlled by a constitutive promoter, pTet-TeR(+LVA) and pTet-TetR(no LVA).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Figure <ins class="diffchange diffchange-inline">4</ins>: Plate containing 200 ng/mL doxycycline plated with <i>E. coli</i> WT and <i>E. coli</i> expressing GFP controlled by a constitutive promoter, pTet-TeR(+LVA) and pTet-TetR(no LVA).</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Figure <del class="diffchange diffchange-inline">1</del>: Plate containing 0 ng/mL doxycycline plated with <i>E. coli</i> WT and <i>E. coli</i> expressing GFP controlled by a constitutive promoter, pTet-TeR(+LVA) and pTet-TetR(no LVA).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Figure <ins class="diffchange diffchange-inline">5</ins>: Plate containing 0 ng/mL doxycycline plated with <i>E. coli</i> WT and <i>E. coli</i> expressing GFP controlled by a constitutive promoter, pTet-TeR(+LVA) and pTet-TetR(no LVA).</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Figure <del class="diffchange diffchange-inline">3</del>: Dose response to doxycycline.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Figure <ins class="diffchange diffchange-inline">6</ins>: Dose response to doxycycline.</div></td></tr>
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</table>Ulrikasimonehttp://2014.igem.org/wiki/index.php?title=Team:SDU-Denmark/Tour40&diff=399636&oldid=prevUlrikasimone at 03:44, 18 October 20142014-10-18T03:44:18Z<p></p>
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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"><span class="intro">OneProt is a self-designed protein</span> containing the correct ratio of essential amino acids and the correct ratio between the essential and non-essential amino acids. Because the protein is self-designed, we wanted to detect if the protein was expressed in <i>E. coli</i> K12 MG1655 and if it stresses the cells and affects their growth. In order to test if the protein is expressed, we made a Western blot, taking advance of the FLAG-tag on OneProt. The Western blot shows that the protein is expressed; however, we cannot tell if the protein has been cut by proteases or not. <br><br></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><a class="popupImg <ins class="diffchange diffchange-inline">alignRight</ins>" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" title="Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. "></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/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" style="width:200px" /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img src="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" style="width:200px" /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></a></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;"><span class="intro">OneProt is a self-designed protein</span> containing the correct ratio of essential amino acids and the correct ratio between the essential and non-essential amino acids. Because the protein is self-designed, we wanted to detect if the protein was expressed in <i>E. coli</i> K12 MG1655 and if it stresses the cells and affects their growth. In order to test if the protein is expressed, we made a Western blot, taking advance of the FLAG-tag on OneProt. The Western blot shows that the protein is expressed; however, we cannot tell if the protein has been cut by proteases or not. <br><br></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><span class="intro">To test if the expression of the protein affects growth-rate</span>, we measured OD on <i>E. coli</i> expressing OneProt using a wild-type as control. The growth curve illustrates that the growth-rate of the cell expressing OneProt isn't affected much compared to that of the wild-type.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><span class="intro">To test if the expression of the protein affects growth-rate</span>, we measured OD on <i>E. coli</i> expressing OneProt using a wild-type as control. The growth curve illustrates that the growth-rate of the cell expressing OneProt isn't affected much compared to that of the wild-type.<br><br></div></td></tr>
</table>Ulrikasimonehttp://2014.igem.org/wiki/index.php?title=Team:SDU-Denmark/Tour40&diff=399518&oldid=prevUlrikasimone at 03:43, 18 October 20142014-10-18T03:43:33Z<p></p>
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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/2014/4/4e/2014SDUWestern_blot_with_GroL.jpg" style="width:400px" /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img src="https://static.igem.org/mediawiki/2014/4/4e/2014SDUWestern_blot_with_GroL.jpg" style="width:400px" /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 1: Western blot showing that the expression of OneProt can be detected. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 1: Western blot showing that the expression of OneProt can be detected. </div></td></tr>
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</table>Ulrikasimonehttp://2014.igem.org/wiki/index.php?title=Team:SDU-Denmark/Tour40&diff=398799&oldid=prevDanie12 at 03:38, 18 October 20142014-10-18T03:38:33Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><span class="intro">In order for us to</span> be able to control the amount of OneProt produced, we need a promoter that can be controlled. For this, we put OneProt under expressional control by pTet. Besides investigating the controllable expression, we also investigated the influence of the LVA tag on TetR. We proved that the inhibition of pTet by TetR is correlated with the concentration of inducer – increasing amounts of inducer means decreasing levels of inhibition of pTet. Comparing the expression by pTet controlled by TetR with and without LVA tag shows that there is a higher expression upon induction, using TetR with LVA than using TetR without LVA. We also show that the pTet is most likely leaky <del class="diffchange diffchange-inline">when </del>when on high-copy plasmids.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><span class="intro">In order for us to</span> be able to control the amount of OneProt produced, we need a promoter that can be controlled. For this, we put OneProt under expressional control by pTet. Besides investigating the controllable expression, we also investigated the influence of the LVA tag on TetR. We proved that the inhibition of pTet by TetR is correlated with the concentration of inducer – increasing amounts of inducer means decreasing levels of inhibition of pTet. Comparing the expression by pTet controlled by TetR with and without LVA tag shows that there is a higher expression upon induction, using TetR with LVA than using TetR without LVA. We also show that the pTet is most likely leaky when on high-copy plasmids.</div></td></tr>
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</table>Danie12http://2014.igem.org/wiki/index.php?title=Team:SDU-Denmark/Tour40&diff=398724&oldid=prevDanie12 at 03:38, 18 October 20142014-10-18T03:38:02Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><span class="intro">To test if the expression of the protein affects growth-rate</span>, we measured OD on <i>E. coli</i> expressing OneProt using a wild-type as control. The growth curve illustrates that the growth-rate of the cell expressing OneProt isn't affected much compared to that of the wild-type.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><span class="intro">To test if the expression of the protein affects growth-rate</span>, we measured OD on <i>E. coli</i> expressing OneProt using a wild-type as control. The growth curve illustrates that the growth-rate of the cell expressing OneProt isn't affected much compared to that of the wild-type.<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><span class="intro">Even though we now know that OneProt is expressed</span> and that <i>E. coli</i> continues to grow, we want to make sure that the protein is not toxic upon digestion. In order for us to do so, we fed <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. To stress <i>C. elegans</i>, and thereby making it more sensitive, we used heat chock assay and stressed the organisms even more after 5 hours. The results are clear: after 7 hours of heat <del class="diffchange diffchange-inline">chock</del>, all <i>C.elegans</i> were alive, and thus we conclude that the OneProt has no toxic effects. <br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><span class="intro">Even though we now know that OneProt is expressed</span> and that <i>E. coli</i> continues to grow, we want to make sure that the protein is not toxic upon digestion. In order for us to do so, we fed <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. To stress <i>C. elegans</i>, and thereby making it more sensitive, we used heat chock assay and stressed the organisms even more after 5 hours. The results are clear: after 7 hours of heat<ins class="diffchange diffchange-inline">-shock assay</ins>, all <i>C.elegans</i> were alive, and thus we conclude that the OneProt has no toxic effects. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="popupImg alignCenter" style="width:300px" target="_blank" title="Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt. "></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="popupImg alignCenter" style="width:300px" target="_blank" title="Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt. "></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:300px" /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:300px" /></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><span class="intro">In order for us to</span> be able to control the amount of OneProt produced, we need a promoter that can be controlled. For this, we put OneProt under expressional control by pTet. Besides investigating the controllable expression, we also investigated the influence of the LVA tag on TetR. We proved that the inhibition of pTet by TetR is correlated with the concentration of inducer – increasing amounts of inducer means decreasing levels of inhibition of pTet. Comparing the expression by pTet controlled by TetR with and without LVA tag shows that there is a higher expression upon induction, using TetR with LVA than using TetR without LVA. We also show that the pTet is most likely leaky.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><span class="intro">In order for us to</span> be able to control the amount of OneProt produced, we need a promoter that can be controlled. For this, we put OneProt under expressional control by pTet. Besides investigating the controllable expression, we also investigated the influence of the LVA tag on TetR. We proved that the inhibition of pTet by TetR is correlated with the concentration of inducer – increasing amounts of inducer means decreasing levels of inhibition of pTet. Comparing the expression by pTet controlled by TetR with and without LVA tag shows that there is a higher expression upon induction, using TetR with LVA than using TetR without LVA. We also show that the pTet is most likely leaky <ins class="diffchange diffchange-inline">when when on high-copy plasmids</ins>.</div></td></tr>
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</table>Danie12http://2014.igem.org/wiki/index.php?title=Team:SDU-Denmark/Tour40&diff=397831&oldid=prevDanie12 at 03:31, 18 October 20142014-10-18T03:31:35Z<p></p>
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<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>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>OneProt is a self-designed protein containing the correct ratio of essential amino acids and the correct ratio between the essential and non-essential amino acids. Because the protein is self-designed, we wanted to detect if the protein was expressed in <i>E. coli</i> K12 MG1655 and if it stresses the cells and affects their growth. In order to test if the protein is expressed, we made a Western blot, taking advance of the FLAG-tag on OneProt. The Western blot shows that the protein is expressed; however, we cannot tell if the protein has been cut by proteases or not. <br><br></div></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"><span class="intro"></ins>OneProt is a self-designed protein<ins class="diffchange diffchange-inline"></span> </ins>containing the correct ratio of essential amino acids and the correct ratio between the essential and non-essential amino acids. Because the protein is self-designed, we wanted to detect if the protein was expressed in <i>E. coli</i> K12 MG1655 and if it stresses the cells and affects their growth. In order to test if the protein is expressed, we made a Western blot, taking advance of the FLAG-tag on OneProt. The Western blot shows that the protein is expressed; however, we cannot tell if the protein has been cut by proteases or not. <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: #eee; color:black; font-size: smaller;"><div><a class="popupImg alignLeft" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" title="Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. "></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a class="popupImg alignLeft" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" title="Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. "></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: #ffa; color:black; font-size: smaller;"><div>To test if the expression of the protein <del class="diffchange diffchange-inline">stresses the cells metabolism</del>, we measured OD on <i>E. coli</i> expressing OneProt using a wild-type as control. The growth curve illustrates that the growth-rate of the cell expressing OneProt isn't affected much compared to that of the wild-type.<br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><span class="intro"></ins>To test if the expression of the protein <ins class="diffchange diffchange-inline">affects growth-rate</span></ins>, we measured OD on <i>E. coli</i> expressing OneProt using a wild-type as control. The growth curve illustrates that the growth-rate of the cell expressing OneProt isn't affected much compared to that of the wild-type.<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>Even though we now know that OneProt is expressed and that <i>E. coli</i> continues to grow, we want to make sure that the protein is not toxic upon digestion. In order for us to do so, we fed <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. To stress <i>C. elegans</i>, and thereby making it more sensitive, we used heat chock assay and stressed the organisms even more after 5 hours. The results are clear: after 7 hours of heat chock, all <i>C.elegans</i> were alive, and thus we conclude that the OneProt has no toxic effects. <br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><span class="intro"></ins>Even though we now know that OneProt is expressed<ins class="diffchange diffchange-inline"></span> </ins>and that <i>E. coli</i> continues to grow, we want to make sure that the protein is not toxic upon digestion. In order for us to do so, we fed <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. To stress <i>C. elegans</i>, and thereby making it more sensitive, we used heat chock assay and stressed the organisms even more after 5 hours. The results are clear: after 7 hours of heat chock, all <i>C.elegans</i> were alive, and thus we conclude that the OneProt has no toxic effects. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="popupImg alignCenter" style="width:300px" target="_blank" title="Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt. "></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="popupImg alignCenter" style="width:300px" target="_blank" title="Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt. "></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:300px" /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:300px" /></div></td></tr>
</table>Danie12http://2014.igem.org/wiki/index.php?title=Team:SDU-Denmark/Tour40&diff=397484&oldid=prevDanie12 at 03:29, 18 October 20142014-10-18T03:29:13Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To test if the expression of the protein stresses the cells metabolism, we measured OD on <i>E. coli</i> expressing OneProt using a wild-type as control. The growth curve illustrates that the growth-rate of the cell isn't affected much compared to that of the wild-type.<br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To test if the expression of the protein stresses the cells metabolism, we measured OD on <i>E. coli</i> expressing OneProt using a wild-type as control. The growth curve illustrates that the growth-rate of the cell <ins class="diffchange diffchange-inline">expressing OneProt </ins>isn't affected much compared to that of the wild-type.<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: #eee; color:black; font-size: smaller;"><div>Even though we now know that OneProt is expressed and that <i>E. coli</i> continues to grow, we want to make sure that the protein is not toxic upon digestion. In order for us to do so, we fed <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. To stress <i>C. elegans</i>, and thereby making it more sensitive, we used heat chock assay and stressed the organisms even more after 5 hours. The results are clear: after 7 hours of heat chock, all <i>C.elegans</i> were alive, and thus we conclude that the OneProt has no toxic effects. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Even though we now know that OneProt is expressed and that <i>E. coli</i> continues to grow, we want to make sure that the protein is not toxic upon digestion. In order for us to do so, we fed <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. To stress <i>C. elegans</i>, and thereby making it more sensitive, we used heat chock assay and stressed the organisms even more after 5 hours. The results are clear: after 7 hours of heat chock, all <i>C.elegans</i> were alive, and thus we conclude that the OneProt has no toxic effects. <br><br></div></td></tr>
</table>Danie12http://2014.igem.org/wiki/index.php?title=Team:SDU-Denmark/Tour40&diff=397343&oldid=prevDanie12 at 03:28, 18 October 20142014-10-18T03:28:22Z<p></p>
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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"><span class="intro"></del>OneProt is a self-designed<del class="diffchange diffchange-inline"></span> </del>protein containing the correct ratio of essential amino acids and the correct ratio between the essential and non-essential amino acids. Because the protein is self-designed, we wanted to detect if the protein was expressed in <i>E. coli</i> K12 MG1655 and if it stresses the cells and affects their growth. In order to test if the protein is expressed, we made a Western blot, taking advance of the FLAG-tag on OneProt. The Western blot shows that the protein is expressed; however, we cannot tell if the protein has been cut by proteases or not. <br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>OneProt is a self-designed protein containing the correct ratio of essential amino acids and the correct ratio between the essential and non-essential amino acids. Because the protein is self-designed, we wanted to detect if the protein was expressed in <i>E. coli</i> K12 MG1655 and if it stresses the cells and affects their growth. In order to test if the protein is expressed, we made a Western blot, taking advance of the FLAG-tag on OneProt. The Western blot shows that the protein is expressed; however, we cannot tell if the protein has been cut by proteases or not. <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: #eee; color:black; font-size: smaller;"><div><a class="popupImg alignLeft" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" title="Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. "></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a class="popupImg alignLeft" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" title="Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. "></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"><span class="intro"></del>To test if the expression<del class="diffchange diffchange-inline"></span> </del>of the protein stresses the cells metabolism, we measured OD on <i>E. coli</i> expressing OneProt using a wild-type as control. The growth curve illustrates that the <del class="diffchange diffchange-inline">metabolism </del>of the cell <del class="diffchange diffchange-inline">is stressed </del>compared to that of the wild-type<del class="diffchange diffchange-inline">, however, the protein is expressed throughout the growth of <i>E. coli</i></del>.<br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To test if the expression of the protein stresses the cells metabolism, we measured OD on <i>E. coli</i> expressing OneProt using a wild-type as control. The growth curve illustrates that the <ins class="diffchange diffchange-inline">growth-rate </ins>of the cell <ins class="diffchange diffchange-inline">isn't affected much </ins>compared to that of the wild-type.<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"><span class="intro"></del>Even though we now<del class="diffchange diffchange-inline"></span> </del>know that OneProt is expressed and that <i>E. coli</i> continues to grow, we want to make sure that the protein is not toxic upon digestion. In for us to do so, we fed <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. To stress C. elegans, and thereby making it more sensitive, we used heat chock assay and stressed the organisms even more after 5 hours. The results are clear: after 7 hours of heat chock, all <i>C.elegans</i> were alive and thus we conclude that the OneProt has no toxic effects. <br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Even though we now know that OneProt is expressed and that <i>E. coli</i> continues to grow, we want to make sure that the protein is not toxic upon digestion. In <ins class="diffchange diffchange-inline">order </ins>for us to do so, we fed <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. To stress <ins class="diffchange diffchange-inline"><i></ins>C. elegans<ins class="diffchange diffchange-inline"></i></ins>, and thereby making it more sensitive, we used heat chock assay and stressed the organisms even more after 5 hours. The results are clear: after 7 hours of heat chock, all <i>C.elegans</i> were alive<ins class="diffchange diffchange-inline">, </ins>and thus we conclude that the OneProt has no toxic effects. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="popupImg alignCenter" style="width:300px" target="_blank" title="Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt. "></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="popupImg alignCenter" style="width:300px" target="_blank" title="Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt. "></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><span class="intro">In order for us to</span> be able to control the amount of OneProt produced, we need a promoter that can be controlled. For this, we put OneProt under expressional control by pTet. Besides investigating the controllable expression, we also investigated the influence of the LVA tag on TetR. We proved that the inhibition of pTet by TetR is correlated with the concentration of inducer – increasing amounts of inducer means decreasing levels of inhibition of pTet. Comparing the expression by pTet controlled by TetR with and without LVA tag shows that there is a higher expression upon induction, using TetR with LVA than using TetR without LVA. We also show that the pTet is leaky.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><span class="intro">In order for us to</span> be able to control the amount of OneProt produced, we need a promoter that can be controlled. For this, we put OneProt under expressional control by pTet. Besides investigating the controllable expression, we also investigated the influence of the LVA tag on TetR. We proved that the inhibition of pTet by TetR is correlated with the concentration of inducer – increasing amounts of inducer means decreasing levels of inhibition of pTet. Comparing the expression by pTet controlled by TetR with and without LVA tag shows that there is a higher expression upon induction, using TetR with LVA than using TetR without LVA. We also show that the pTet is <ins class="diffchange diffchange-inline">most likely </ins>leaky.</div></td></tr>
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</table>Danie12http://2014.igem.org/wiki/index.php?title=Team:SDU-Denmark/Tour40&diff=395797&oldid=prevSarahNielsen at 03:19, 18 October 20142014-10-18T03:19:43Z<p></p>
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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 03:19, 18 October 2014</td>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>OneProt is a self-designed protein containing the correct ratio of essential amino acids and the correct ratio between the essential and non-essential amino acids. Because the protein is self-designed, we wanted to detect if the protein was expressed in <i>E. coli</i> K12 MG1655 and if it stresses the cells and affects their growth. In order to test if the protein is expressed, we made a Western blot, taking advance of the FLAG-tag on OneProt. The Western blot shows that the protein is expressed; however, we cannot tell if the protein has been cut by proteases or not. <br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><span class="intro"></ins>OneProt is a self-designed<ins class="diffchange diffchange-inline"></span> </ins>protein containing the correct ratio of essential amino acids and the correct ratio between the essential and non-essential amino acids. Because the protein is self-designed, we wanted to detect if the protein was expressed in <i>E. coli</i> K12 MG1655 and if it stresses the cells and affects their growth. In order to test if the protein is expressed, we made a Western blot, taking advance of the FLAG-tag on OneProt. The Western blot shows that the protein is expressed; however, we cannot tell if the protein has been cut by proteases or not. <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: #eee; color:black; font-size: smaller;"><div><a class="popupImg alignLeft" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" title="Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. "></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a class="popupImg alignLeft" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" title="Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. "></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To test if the expression of the protein stresses the cells metabolism, we measured OD on <i>E. coli</i> expressing OneProt using a wild-type as control. The growth curve illustrates that the metabolism of the cell is stressed compared to that of the wild-type, however, the protein is expressed throughout the growth of <i>E. coli</i>.<br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><span class="intro"></ins>To test if the expression<ins class="diffchange diffchange-inline"></span> </ins>of the protein stresses the cells metabolism, we measured OD on <i>E. coli</i> expressing OneProt using a wild-type as control. The growth curve illustrates that the metabolism of the cell is stressed compared to that of the wild-type, however, the protein is expressed throughout the growth of <i>E. coli</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>Even though we now know that OneProt is expressed and that <i>E. coli</i> continues to grow, we want to make sure that the protein is not toxic upon digestion. In for us to do so, we fed <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. To stress C. elegans, and thereby making it more sensitive, we used heat chock assay and stressed the organisms even more after 5 hours. The results are clear: after 7 hours of heat chock, all <i>C.elegans</i> were alive and thus we conclude that the OneProt has no toxic effects. <br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><span class="intro"></ins>Even though we now<ins class="diffchange diffchange-inline"></span> </ins>know that OneProt is expressed and that <i>E. coli</i> continues to grow, we want to make sure that the protein is not toxic upon digestion. In for us to do so, we fed <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. To stress C. elegans, and thereby making it more sensitive, we used heat chock assay and stressed the organisms even more after 5 hours. The results are clear: after 7 hours of heat chock, all <i>C.elegans</i> were alive and thus we conclude that the OneProt has no toxic effects. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="popupImg alignCenter" style="width:300px" target="_blank" title="Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt. "></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="popupImg alignCenter" style="width:300px" target="_blank" title="Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt. "></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:300px" /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:300px" /></div></td></tr>
</table>SarahNielsen