http://2014.igem.org/wiki/index.php?title=Special:Contributions/Danie12&feed=atom&limit=50&target=Danie12&year=&month=2014.igem.org - User contributions [en]2024-03-28T09:56:31ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:SDU-Denmark/Tour40Team:SDU-Denmark/Tour402014-10-18T03:38:33Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Results</h3><br />
<br />
<p class='intro'><br />
<font color="3397FE">"I know that I am intelligent, because I know that I know nothing." – <b>Socrates</b></font><br />
</p><br />
<br />
<br />
<p><br />
<br />
<span class="intro">The next few pages will</span> guide you through the results of the characterization of our submitted parts. On this page, you will find short descriptions of our results, leading you to the final result. We hope you will dig deeper into the results of our systems. After the need to prioritize subprojects, the aim of our project was to get <i>E. coli</i> to express a self-designed nutritional protein, controlled by an inducible promoter. Furthermore it will express limonene synthase for the synthesis of limonene, the main part of lemon flavor.<br><br><br />
</p><br />
<br />
<h4>Synthesis of self-designed protein OneProt</h4><br><br />
<div class="popupImg alignCenter" style="width:400px"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4e/2014SDUWestern_blot_with_GroL.jpg" style="width:400px" /><br />
Figure 1: Western blot showing that the expression of OneProt can be detected. <br />
</div><br />
<p><br />
<br />
<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><br />
<br />
<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. "><br />
<img src="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" style="width:200px" /><br />
Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. <br />
</a><br />
<br />
<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><br />
<br />
<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-shock assay, all <i>C.elegans</i> were alive, and thus we conclude that the OneProt has no toxic effects. <br><br><br />
<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. "><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:300px" /><br />
Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt.<br />
</div><br />
<br><br />
</p><br />
<br />
<h4>Controlling the Tet promoter</h4><br />
<p><br />
<a class="popupImg alignRight" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/4f/2014SDUresults5.png" title="Figure 2: 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)."><br />
<img src="https://static.igem.org/mediawiki/2014/1/19/2014SDUresults6.png" style="width:200px" /><br />
Figure 2: 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).<br />
</a><br />
<a class="popupImg alignRight" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b0/2014SDUresults3.png" title="Figure 1: 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)."><br />
<img src="https://static.igem.org/mediawiki/2014/e/e6/2014SDUresults4.png" style="width:200px" /><br />
Figure 1: 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).<br />
</a><br />
<br />
<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.<br />
</p><br />
<br><br><br><br><br />
<div><br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/12/2014SDUresults2.png" title="Figure 3: Dose response to doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/12/2014SDUresults2.png" style="width:500px" /><br />
Figure 3: Dose response to doxycycline.<br />
</a><br><br />
</div><br />
<br />
<h4>Flavor improvement</h4><br />
<p><br />
<span class="intro">The thought of eating <i>E. coli</i></span> does not sound that delicious – and that is why we want our OneProt to taste like lemon. Though the cloning proved unsuccessful. However, we have characterised an odor-free <i>E. coli</i> strain and compared it with a wild type K12 MG1655 <i>E. coli</i> strain. We have used Ion Mobility Spectroscopy to analyse the two strains, since we deemed it among the best methods to analyse odors. From the results it was shown that the odor-free strain does not produce indole, which is the compound responsible for the characteristic odor of <i>E. coli</i>. <br />
<br><br><br />
</p><br />
<br />
<h4>Added parts and devices</h4><br />
<p><br />
<span class="intro">To the great iGEM Registry of Standard Biological Parts</span> we have added 3 basic parts, 2 regulatory devices, 1 constitutively active production device and 4 regulable production devices.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour40Team:SDU-Denmark/Tour402014-10-18T03:38:02Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Results</h3><br />
<br />
<p class='intro'><br />
<font color="3397FE">"I know that I am intelligent, because I know that I know nothing." – <b>Socrates</b></font><br />
</p><br />
<br />
<br />
<p><br />
<br />
<span class="intro">The next few pages will</span> guide you through the results of the characterization of our submitted parts. On this page, you will find short descriptions of our results, leading you to the final result. We hope you will dig deeper into the results of our systems. After the need to prioritize subprojects, the aim of our project was to get <i>E. coli</i> to express a self-designed nutritional protein, controlled by an inducible promoter. Furthermore it will express limonene synthase for the synthesis of limonene, the main part of lemon flavor.<br><br><br />
</p><br />
<br />
<h4>Synthesis of self-designed protein OneProt</h4><br><br />
<div class="popupImg alignCenter" style="width:400px"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4e/2014SDUWestern_blot_with_GroL.jpg" style="width:400px" /><br />
Figure 1: Western blot showing that the expression of OneProt can be detected. <br />
</div><br />
<p><br />
<br />
<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><br />
<br />
<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. "><br />
<img src="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" style="width:200px" /><br />
Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. <br />
</a><br />
<br />
<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><br />
<br />
<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-shock assay, all <i>C.elegans</i> were alive, and thus we conclude that the OneProt has no toxic effects. <br><br><br />
<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. "><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:300px" /><br />
Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt.<br />
</div><br />
<br><br />
</p><br />
<br />
<h4>Controlling the Tet promoter</h4><br />
<p><br />
<a class="popupImg alignRight" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/4f/2014SDUresults5.png" title="Figure 2: 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)."><br />
<img src="https://static.igem.org/mediawiki/2014/1/19/2014SDUresults6.png" style="width:200px" /><br />
Figure 2: 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).<br />
</a><br />
<a class="popupImg alignRight" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b0/2014SDUresults3.png" title="Figure 1: 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)."><br />
<img src="https://static.igem.org/mediawiki/2014/e/e6/2014SDUresults4.png" style="width:200px" /><br />
Figure 1: 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).<br />
</a><br />
<br />
<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 when on high-copy plasmids.<br />
</p><br />
<br><br><br><br><br />
<div><br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/12/2014SDUresults2.png" title="Figure 3: Dose response to doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/12/2014SDUresults2.png" style="width:500px" /><br />
Figure 3: Dose response to doxycycline.<br />
</a><br><br />
</div><br />
<br />
<h4>Flavor improvement</h4><br />
<p><br />
<span class="intro">The thought of eating <i>E. coli</i></span> does not sound that delicious – and that is why we want our OneProt to taste like lemon. Though the cloning proved unsuccessful. However, we have characterised an odor-free <i>E. coli</i> strain and compared it with a wild type K12 MG1655 <i>E. coli</i> strain. We have used Ion Mobility Spectroscopy to analyse the two strains, since we deemed it among the best methods to analyse odors. From the results it was shown that the odor-free strain does not produce indole, which is the compound responsible for the characteristic odor of <i>E. coli</i>. <br />
<br><br><br />
</p><br />
<br />
<h4>Added parts and devices</h4><br />
<p><br />
<span class="intro">To the great iGEM Registry of Standard Biological Parts</span> we have added 3 basic parts, 2 regulatory devices, 1 constitutively active production device and 4 regulable production devices.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour40Team:SDU-Denmark/Tour402014-10-18T03:31:35Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Results</h3><br />
<br />
<p class='intro'><br />
<font color="3397FE">"I know that I am intelligent, because I know that I know nothing." – <b>Socrates</b></font><br />
</p><br />
<br />
<br />
<p><br />
<br />
<span class="intro">The next few pages will</span> guide you through the results of the characterization of our submitted parts. On this page, you will find short descriptions of our results, leading you to the final result. We hope you will dig deeper into the results of our systems. After the need to prioritize subprojects, the aim of our project was to get <i>E. coli</i> to express a self-designed nutritional protein, controlled by an inducible promoter. Furthermore it will express limonene synthase for the synthesis of limonene, the main part of lemon flavor.<br><br><br />
</p><br />
<br />
<h4>Synthesis of self-designed protein OneProt</h4><br><br />
<div class="popupImg alignCenter" style="width:400px"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4e/2014SDUWestern_blot_with_GroL.jpg" style="width:400px" /><br />
Figure 1: Western blot showing that the expression of OneProt can be detected. <br />
</div><br />
<p><br />
<br />
<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><br />
<br />
<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. "><br />
<img src="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" style="width:200px" /><br />
Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. <br />
</a><br />
<br />
<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><br />
<br />
<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 chock, all <i>C.elegans</i> were alive, and thus we conclude that the OneProt has no toxic effects. <br><br><br />
<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. "><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:300px" /><br />
Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt.<br />
</div><br />
<br><br />
</p><br />
<br />
<h4>Controlling the Tet promoter</h4><br />
<p><br />
<a class="popupImg alignRight" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/4f/2014SDUresults5.png" title="Figure 2: 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)."><br />
<img src="https://static.igem.org/mediawiki/2014/1/19/2014SDUresults6.png" style="width:200px" /><br />
Figure 2: 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).<br />
</a><br />
<a class="popupImg alignRight" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b0/2014SDUresults3.png" title="Figure 1: 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)."><br />
<img src="https://static.igem.org/mediawiki/2014/e/e6/2014SDUresults4.png" style="width:200px" /><br />
Figure 1: 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).<br />
</a><br />
<br />
<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.<br />
</p><br />
<br><br><br><br><br />
<div><br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/12/2014SDUresults2.png" title="Figure 3: Dose response to doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/12/2014SDUresults2.png" style="width:500px" /><br />
Figure 3: Dose response to doxycycline.<br />
</a><br><br />
</div><br />
<br />
<h4>Flavor improvement</h4><br />
<p><br />
<span class="intro">The thought of eating <i>E. coli</i></span> does not sound that delicious – and that is why we want our OneProt to taste like lemon. Though the cloning proved unsuccessful. However, we have characterised an odor-free <i>E. coli</i> strain and compared it with a wild type K12 MG1655 <i>E. coli</i> strain. We have used Ion Mobility Spectroscopy to analyse the two strains, since we deemed it among the best methods to analyse odors. From the results it was shown that the odor-free strain does not produce indole, which is the compound responsible for the characteristic odor of <i>E. coli</i>. <br />
<br><br><br />
</p><br />
<br />
<h4>Added parts and devices</h4><br />
<p><br />
<span class="intro">To the great iGEM Registry of Standard Biological Parts</span> we have added 3 basic parts, 2 regulatory devices, 1 constitutively active production device and 4 regulable production devices.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour40Team:SDU-Denmark/Tour402014-10-18T03:29:13Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Results</h3><br />
<br />
<p class='intro'><br />
<font color="3397FE">"I know that I am intelligent, because I know that I know nothing." – <b>Socrates</b></font><br />
</p><br />
<br />
<br />
<p><br />
<br />
<span class="intro">The next few pages will</span> guide you through the results of the characterization of our submitted parts. On this page, you will find short descriptions of our results, leading you to the final result. We hope you will dig deeper into the results of our systems. After the need to prioritize subprojects, the aim of our project was to get <i>E. coli</i> to express a self-designed nutritional protein, controlled by an inducible promoter. Furthermore it will express limonene synthase for the synthesis of limonene, the main part of lemon flavor.<br><br><br />
</p><br />
<br />
<h4>Synthesis of self-designed protein OneProt</h4><br><br />
<div class="popupImg alignCenter" style="width:400px"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4e/2014SDUWestern_blot_with_GroL.jpg" style="width:400px" /><br />
Figure 1: Western blot showing that the expression of OneProt can be detected. <br />
</div><br />
<p><br />
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><br />
<br />
<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. "><br />
<img src="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" style="width:200px" /><br />
Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. <br />
</a><br />
<br />
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 expressing OneProt isn't affected much compared to that of the wild-type.<br><br><br />
<br />
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><br />
<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. "><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:300px" /><br />
Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt.<br />
</div><br />
<br><br />
</p><br />
<br />
<h4>Controlling the Tet promoter</h4><br />
<p><br />
<a class="popupImg alignRight" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/4f/2014SDUresults5.png" title="Figure 2: 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)."><br />
<img src="https://static.igem.org/mediawiki/2014/1/19/2014SDUresults6.png" style="width:200px" /><br />
Figure 2: 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).<br />
</a><br />
<a class="popupImg alignRight" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b0/2014SDUresults3.png" title="Figure 1: 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)."><br />
<img src="https://static.igem.org/mediawiki/2014/e/e6/2014SDUresults4.png" style="width:200px" /><br />
Figure 1: 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).<br />
</a><br />
<br />
<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.<br />
</p><br />
<br><br><br><br><br />
<div><br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/12/2014SDUresults2.png" title="Figure 3: Dose response to doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/12/2014SDUresults2.png" style="width:500px" /><br />
Figure 3: Dose response to doxycycline.<br />
</a><br><br />
</div><br />
<br />
<h4>Flavor improvement</h4><br />
<p><br />
<span class="intro">The thought of eating <i>E. coli</i></span> does not sound that delicious – and that is why we want our OneProt to taste like lemon. Though the cloning proved unsuccessful. However, we have characterised an odor-free <i>E. coli</i> strain and compared it with a wild type K12 MG1655 <i>E. coli</i> strain. We have used Ion Mobility Spectroscopy to analyse the two strains, since we deemed it among the best methods to analyse odors. From the results it was shown that the odor-free strain does not produce indole, which is the compound responsible for the characteristic odor of <i>E. coli</i>. <br />
<br><br><br />
</p><br />
<br />
<h4>Added parts and devices</h4><br />
<p><br />
<span class="intro">To the great iGEM Registry of Standard Biological Parts</span> we have added 3 basic parts, 2 regulatory devices, 1 constitutively active production device and 4 regulable production devices.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour40Team:SDU-Denmark/Tour402014-10-18T03:28:22Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Results</h3><br />
<br />
<p class='intro'><br />
<font color="3397FE">"I know that I am intelligent, because I know that I know nothing." – <b>Socrates</b></font><br />
</p><br />
<br />
<br />
<p><br />
<br />
<span class="intro">The next few pages will</span> guide you through the results of the characterization of our submitted parts. On this page, you will find short descriptions of our results, leading you to the final result. We hope you will dig deeper into the results of our systems. After the need to prioritize subprojects, the aim of our project was to get <i>E. coli</i> to express a self-designed nutritional protein, controlled by an inducible promoter. Furthermore it will express limonene synthase for the synthesis of limonene, the main part of lemon flavor.<br><br><br />
</p><br />
<br />
<h4>Synthesis of self-designed protein OneProt</h4><br><br />
<div class="popupImg alignCenter" style="width:400px"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4e/2014SDUWestern_blot_with_GroL.jpg" style="width:400px" /><br />
Figure 1: Western blot showing that the expression of OneProt can be detected. <br />
</div><br />
<p><br />
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><br />
<br />
<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. "><br />
<img src="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" style="width:200px" /><br />
Figure 2: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 expressing OneProt using a wild-type as control. <br />
</a><br />
<br />
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><br />
<br />
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><br />
<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. "><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:300px" /><br />
Figure 3: <i>C.elegans</i> fed with <i>E. coli</i> K12 MG1655 expressing OneProt.<br />
</div><br />
<br><br />
</p><br />
<br />
<h4>Controlling the Tet promoter</h4><br />
<p><br />
<a class="popupImg alignRight" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/4f/2014SDUresults5.png" title="Figure 2: 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)."><br />
<img src="https://static.igem.org/mediawiki/2014/1/19/2014SDUresults6.png" style="width:200px" /><br />
Figure 2: 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).<br />
</a><br />
<a class="popupImg alignRight" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b0/2014SDUresults3.png" title="Figure 1: 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)."><br />
<img src="https://static.igem.org/mediawiki/2014/e/e6/2014SDUresults4.png" style="width:200px" /><br />
Figure 1: 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).<br />
</a><br />
<br />
<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.<br />
</p><br />
<br><br><br><br><br />
<div><br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/12/2014SDUresults2.png" title="Figure 3: Dose response to doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/12/2014SDUresults2.png" style="width:500px" /><br />
Figure 3: Dose response to doxycycline.<br />
</a><br><br />
</div><br />
<br />
<h4>Flavor improvement</h4><br />
<p><br />
<span class="intro">The thought of eating <i>E. coli</i></span> does not sound that delicious – and that is why we want our OneProt to taste like lemon. Though the cloning proved unsuccessful. However, we have characterised an odor-free <i>E. coli</i> strain and compared it with a wild type K12 MG1655 <i>E. coli</i> strain. We have used Ion Mobility Spectroscopy to analyse the two strains, since we deemed it among the best methods to analyse odors. From the results it was shown that the odor-free strain does not produce indole, which is the compound responsible for the characteristic odor of <i>E. coli</i>. <br />
<br><br><br />
</p><br />
<br />
<h4>Added parts and devices</h4><br />
<p><br />
<span class="intro">To the great iGEM Registry of Standard Biological Parts</span> we have added 3 basic parts, 2 regulatory devices, 1 constitutively active production device and 4 regulable production devices.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour42Team:SDU-Denmark/Tour422014-10-18T03:05:31Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>OneProt</h3><br />
<p><br />
<br />
<span class="intro">The pTet expression system</span> and limonene synthase construct is evolved around one thing: the OneProt.<br />
We have made the pTet-OneProt construct in order for us to synthesize a nutritional protein with the <br />
correct ratio of essential amino acids and the correct ratio between essential and non-essential <br />
amino acids. The device is found as <a href="http://parts.igem.org/Part:BBa_K1475000"> Bba_K1475000.</a><br><br><br />
<br />
<span class="intro">The protein is self-designed,</span> so we wanted to test if the protein were expressed in <i>E. coli</i> K12 MG1655, by<br />
the use of Western blotting. The western blot was blottet with <i>E. coli</i> K12 MG1655 wild-type and <i>E. coli</i><br />
expressing OneProt at different OD measures.<br><br><br />
</p><br />
<br />
<div class="popupImg alignCenter" style="width:500px"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4e/2014SDUWestern_blot_with_GroL.jpg" style="width:500px" /><br />
Figure 1: Western blot showing <i>E. coli</i> wild-type at OD600 at 0.3, 0.8 and 1.8 and <i>E. coli</i> expressing OneProt at OD600 at 0.3, 0.8, 1.8 and an overnight culture. <br />
</div><br />
<br />
<p><br />
<br><br />
<span class="intro">The protein has a</span> 3xFLAG tag and since bonds are showing, OneProt is expressed. However, from this<br />
western blot, we cannot see if the protein has been cut, just that it is expressed.<br />
</p><br />
<p><br />
<a class="popupImg alignRight" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" title="Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control."><br />
<img src="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" style="width:250px" /><br />
Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control.<br />
</a><br />
<br />
In order to check that we had the protein expressed in its full length, we did a coomassie stain on a SDS-<br />
page. Here we also wanted to receive information on the expression of the protein at different growth <br />
stages of <i>E. coli</i>. We analyzed samples from early exponential phase (OD600=0.3), late exponential phase <br />
(OD600=1.5), stationary phase (OD=2.5) and an overnight culture. As a control, <i>E. coli</i> with an empty vector <br />
(PSC1C3) was used.<br><br><br />
<br />
<span class="intro">OneProt has a molecular</span> weight of approximately 53.7 kDa. Unfortunately, there is no clear bond at this length. However, there is a bond at approximately 25 kDa, which is not detected in the control. We cannot <br />
conclude what gives rise to the band, but it might be a cellular response to an unfolded protein.<br><br><br />
<br />
<span class="intro">To test what effect</span> the expression of OneProt have on <i>E. coli</i> we set up a growth experiment<br />
measuring OD over time on the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 <br />
expressing OneProt and with an empty vector.<br><br><br />
<br />
</p><br />
<p><br />
<a class="popupImg alignLeft" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" title="Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector."><br />
<img src="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" style="width:250px" /><br />
Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector.<br />
</a><br />
<br />
From the growth curve, it is shown that the growth-rate of neither the strain expressing the OneProt protein nor the YYC912 strain is affected a lot compared to the <i>E. coli</i> K12 wild-type.<br><br><br />
<br />
<span class="intro">By comparing the growth curve</span> of <i>E. coli</i> K12 expressing OneProt, TetR(+LVA), TetR(no LVA) and odor-free <i>E. coli</i> YYC912 it is seen that the growth-rate of <i>E. coli</i> expressing TetR is lowered compared to <br />
the other strains, which means that it might be difficult to have OneProt expressed in high amounts controlled <br />
by pTet (+/-LVA), with the TetR repressors overexpressed at present levels. However, as argued on the previous page, the levels of TetR would have to be lowered, which would probably alleviate the attenuated growth-rate. <br><br><br />
</p><br />
<p><br />
<a class="popupImg alignRight" style="width:400px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" title="Figure 4: Growth curves showing <i>E. coli</i> K12 MG 1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:400px" /><br />
Figure 4: Growth curves showing <i>E. coli</i> K12 MG1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912.<br />
</a><br />
<br />
<span class="intro">Because OneProt is self-designed</span>, we wanted to test if the protein has any toxicity. To do so, we fed <br />
<i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector <br />
expressing OneProt on separate plates. On both plates, 20 <i>C. elegans</i> were tested. Articles recommend <br />
using heat shock assay for 7 hours: 1 hour at 35° C followed by 1 hour at 22°C, <br />
<span class="sourceReference">repeated.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Mosbech, M., et al.: Functional Loss of Two Ceramide Synthases Elicits Autophagy-Dependent Lifespan Extension in C. <br />
elegans.: PLoS ONE, 2013. 8 vol:7.<br />
<br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/23894595" target="_blank">(Link)</a></span><br />
<span class="sourceReference">&nbsp;</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Rodriguez, M., et al.:Worms under stress: <i>C. elegans</i> stress response and its relevance to complex human disease and <br />
aging. Trends in Genetics, 2013. Vol: 29, 6, p. 367-374.<br />
<br />
<a href="http://www.sciencedirect.com/science/article/pii/S016895251300022X" target="_blank">(Link)</a></span><br><br><br />
<br />
<span class="intro">After approximately 5 hours</span> no effects on <i>C. elegans</i> was detectable. Therefore we decided to stress <br />
<i>C. elegans</i> a little more, incubating them in 2 hours at 35°C followed by 1 hour at 22°C, repeated. After 7 <br />
hours, every <i>C. elegans</i> on both plates were alive. Thus we conclude that the strain expressing OneProt at presents levels doesn't display toxic effects on <i>C. elegans</i>. This suggest that there are no obvious toxic effects towards eukaryotic cells such as human cells. However, the resulst are far from conclusive, and extensive testing is necessarry to confirm this suggestion.<br><br><br />
</p><br />
<br />
<div class="popupImg alignCenter" style="width:400px" target="_blank" title="Figure 5:Picture of C. elegans fed with <i>E. coli</i> K12 MG1655 expressing OneProt."><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:400px" /><br />
Figure 5: Picture of C. elegans fed with <i>E. coli</i> K12 MG1655 expressing OneProt.<br />
</div><br />
<br><br><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour42Team:SDU-Denmark/Tour422014-10-18T02:58:12Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>OneProt</h3><br />
<p><br />
<br />
<span class="intro">The pTet expression system</span> and limonene synthase construct is evolved around one thing: the OneProt.<br />
We have made the pTet-OneProt construct in order for us to synthesize a nutritional protein with the <br />
correct ratio of essential amino acids and the correct ratio between essential and non-essential <br />
amino acids. The device is found as <a href="http://parts.igem.org/Part:BBa_K1475000"> Bba_K1475000.</a><br><br><br />
<br />
<span class="intro">The protein is self-designed,</span> so we wanted to test if the protein were expressed in <i>E. coli</i> K12 MG1655, by<br />
the use of Western blotting. The western blot was blottet with <i>E. coli</i> K12 MG1655 wild-type and <i>E. coli</i><br />
expressing OneProt at different OD measures.<br><br><br />
</p><br />
<br />
<div class="popupImg alignCenter" style="width:500px"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4e/2014SDUWestern_blot_with_GroL.jpg" style="width:500px" /><br />
Figure 1: Western blot showing <i>E. coli</i> wild-type at OD600 at 0.3, 0.8 and 1.8 and <i>E. coli</i> expressing OneProt at OD600 at 0.3, 0.8, 1.8 and an overnight culture. <br />
</div><br />
<br />
<p><br />
<br><br />
<span class="intro">The protein has a</span> 3xFLAG tag and since bonds are showing, OneProt is expressed. However, from this<br />
western blot, we cannot see if the protein has been cut, just that it is expressed.<br />
</p><br />
<p><br />
<a class="popupImg alignRight" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" title="Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control."><br />
<img src="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" style="width:250px" /><br />
Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control.<br />
</a><br />
<br />
In order to check that we had the protein expressed in its full length, we did a coomassie stain on a SDS-<br />
page. Here we also wanted to receive information on the expression of the protein at different growth <br />
stages of <i>E. coli</i>. We analyzed samples from early exponential phase (OD600=0.3), late exponential phase <br />
(OD600=1.5), stationary phase (OD=2.5) and an overnight culture. As a control, <i>E. coli</i> with an empty vector <br />
(PSC1C3) was used.<br><br><br />
<br />
<span class="intro">OneProt has a molecular</span> weight of approximately 53.7 kDa. Unfortunately, there is no clear bond at this length. However, there is a bond at approximately 25 kDa, which is not detected in the control. We cannot <br />
conclude what gives rise to the band, but it might be a cellular response to an unfolded protein.<br><br><br />
<br />
<span class="intro">To test what effect</span> the expression of OneProt have on <i>E. coli</i> we set up a growth experiment<br />
measuring OD over time on the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 <br />
expressing OneProt and with an empty vector.<br><br><br />
<br />
</p><br />
<p><br />
<a class="popupImg alignLeft" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" title="Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector."><br />
<img src="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" style="width:250px" /><br />
Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector.<br />
</a><br />
<br />
From the growth curve, it is shown that the growth-rate of neither the strain expressing the OneProt protein nor the YYC912 strain is affected a lot compared to the <i>E. coli</i> K12 wild-type.<br><br><br />
<br />
<span class="intro">By comparing the growth curve</span> of <i>E. coli</i> K12 expressing OneProt, TetR(+LVA), TetR(no LVA) and odor-free <i>E. coli</i> YYC912 it is seen that the growth-rate of <i>E. coli</i> expressing TetR is lowered compared to <br />
the other strains, which means that it might be difficult to have OneProt expressed in high amounts controlled <br />
by pTet (+/-LVA), with the TetR repressors overexpressed at present levels. However, as argued on previous page, the levels of TetR would have to be lowered, which would probably alleviate the attenuated growth-rate. <br><br><br />
</p><br />
<p><br />
<a class="popupImg alignRight" style="width:400px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" title="Figure 4: Growth curves showing <i>E. coli</i> K12 MG 1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:400px" /><br />
Figure 4: Growth curves showing <i>E. coli</i> K12 MG1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912.<br />
</a><br />
<br />
Because OneProt is self-designed, we wanted to test if the protein has any toxicity. To do so, we fed <br />
<i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector <br />
expressing OneProt on separate plates. On both plates, 20 <i>C. elegans</i> were tested. Articles recommend <br />
using heat shock assay for 7 hours: 1 hour at 35° C followed by 1 hour at 22°C, <br />
<span class="sourceReference">repeated.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Mosbech, M., et al.: Functional Loss of Two Ceramide Synthases Elicits Autophagy-Dependent Lifespan Extension in C. <br />
elegans.: PLoS ONE, 2013. 8 vol:7.<br />
<br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/23894595" target="_blank">(Link)</a></span><br />
<span class="sourceReference">&nbsp;</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Rodriguez, M., et al.:Worms under stress: <i>C. elegans</i> stress response and its relevance to complex human disease and <br />
aging. Trends in Genetics, 2013. Vol: 29, 6, p. 367-374.<br />
<br />
<a href="http://www.sciencedirect.com/science/article/pii/S016895251300022X" target="_blank">(Link)</a></span><br><br><br />
<br />
<span class="intro">After approximately 5 hours</span> no effects on <i>C. elegans</i> was detectable. Therefore we decided to stress <br />
<i>C. elegans</i> a little more, incubating them in 2 hours at 35°C followed by 1 hour at 22°C, repeated. After 7 <br />
hours, every <i>C. elegans</i> on both plates were alive. Thus we conclude that the protein has no toxic effect.<br><br><br />
</p><br />
<br />
<div class="popupImg alignCenter" style="width:400px" target="_blank" title="Figure 5:Picture of C. elegans fed with <i>E. coli</i> K12 MG1655 expressing OneProt."><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:400px" /><br />
Figure 5: Picture of C. elegans fed with <i>E. coli</i> K12 MG1655 expressing OneProt.<br />
</div><br />
<br><br><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-18T02:38:49Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">As mentioned on the System design page</span>, we wanted to investigate the Tet promoter expression profile, and what influence the LVA tag on the pTet promoter regulator (TetR) had on the expression profile. <br><br><br />
<br />
<span class="intro">For this purpose</span> three plasmids were constructed. The first expressing GFP from pTet promoter with no regulation. The second and third constitutively expressing TetR with or without the LVA tag as well<br><br><br />
<br />
<span class="intro">The pTet-GFP construct</span> was cloned.<br><br><br />
<br />
<span class="intro">A TetR (no LVA) construct</span> was cloned by PCR amplification without the LVA tag and addition of promoter, RBS and terminator. Subsequently the construct was ligated into the pTet-GFP construct. The constructs can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475004" target="_blank">Bba_K1475004</a> and <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>, respectively.<br><br><br />
<br />
<span class="intro">A TetR (with LVA) construct</span> was cloned by PCR amplification with the LVA tag and addition of the same promoter, RBS and terminator as TetR without LVA tag. Subsequently the construct was ligated into the pTet-GFP construct, as well. The construct can be found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in</span> the TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
<span class="intro">To test if the Tet promoter</span> could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro"> Comparing only the strains expressing either variants of TetR</span>, the results of the FACS illustrates that without induction with doxycycline, GFP is still expressed. Most likely because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduced in the constructs containing TetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Although the FACS results indicates</span> that the pTet inhibited by TetR with LVA tag is the most responsive upon induction by doxycycline, we argue that the effect seen is due to overexpressing of TetR repressor. The hypothesis is based on the poor median fluorescence compared to un-regulated pTet promoter, even at doxycycline concentrations inhibitory of cell growth. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins. This could explain the better response from induction of TetR with LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without, supporting this hypothesis.<br><br><br />
<br />
<span class = "intro">By using a strain,</span> constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer, futher supporting the hypothesis. <br />
<br><br><br />
<br />
<span class = "intro">To analyse the amount</span> of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
<span class = "intro">Because pTet is leaky,</span> all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with doxycycline</span> were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<span class = "intro">Table 1:</span> Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
<br><br />
<span class="intro">Table 2:</span><br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP constructs</span> on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it was also seen from the FACS <br />
results. Furthermore, the plating assay proves that the bricks are functional, however slowly responding to induction (continuous induction over 24 hours compared to induction over 1 hour)<br><br><br />
<br />
<span class="intro">To see how the growth</span> of the bacteria expressing GFP controlled by pTet are affected, we measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the growth</span> of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team proved</span> that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-18T02:37:27Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">As mentioned on the System design page</span>, we wanted to investigate the Tet promoter expression profile, and what influence the LVA tag on the pTet promoter regulator (TetR) had on the expression profile. <br><br><br />
<br />
<span class="intro">For this purpose</span> three plasmids were constructed. The first expressing GFP from pTet promoter with no regulation. The second and third constitutively expressing TetR with or without the LVA tag as well<br><br><br />
<br />
<span class="intro">The pTet-GFP construct</span> was cloned.<br><br><br />
<br />
<span class="intro">A TetR (no LVA) construct</span> was cloned by PCR amplification without the LVA tag and addition of promoter, RBS and terminator. Subsequently the construct was ligated into the pTet-GFP construct. The constructs can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475004" target="_blank">Bba_K1475004</a> and <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>, respectively.<br><br><br />
<br />
<span class="intro">A TetR (with LVA) construct</span> was cloned by PCR amplification with the LVA tag and addition of the same promoter, RBS and terminator as TetR without LVA tag. Subsequently the construct was ligated into the pTet-GFP construct, as well. The construct can be found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in</span> the TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
<span class="intro">To test if the Tet promoter</span> could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro"> Comparing only the strains expressing either variants of TetR</span>, the results of the FACS illustrates that without induction with doxycycline, GFP is still expressed. Most likely because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduced in the constructs containing TetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Although the FACS results indicates</span> that the pTet inhibited by TetR with LVA tag is the most responsive upon induction by doxycycline, we argue that the effect seen is due to overexpressing of TetR repressor. The hypothesis is based on the poor median fluorescence compared to un-regulated pTet promoter, even at doxycycline concentrations inhibitory of cell growth. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins. This could explain the better response from induction of TetR with LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without supporting this hypothesis.<br><br><br />
<br />
<span class = "intro">By using a strain,</span> constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer, futher supporting the hypothesis. <br />
<br><br><br />
<br />
<span class = "intro">To analyse the amount</span> of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
<span class = "intro">Because pTet is leaky,</span> all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with doxycycline</span> were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<span class = "intro">Table 1:</span> Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
<br><br />
<span class="intro">Table 2:</span><br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP constructs</span> on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it was also seen from the FACS <br />
results. Furthermore, the plating assay proves that the bricks are functional, however slowly responding to induction (continuous induction over 24 hours compared to induction over 1 hour)<br><br><br />
<br />
<span class="intro">To see how the growth</span> of the bacteria expressing GFP controlled by pTet are affected, we measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the growth</span> of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team proved</span> that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-18T02:23:47Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">As mentioned on the System design page</span>, we wanted to investigate the Tet promoter expression profile, and what influence the LVA tag on the pTet promoter regulator (TetR) had on the expression profile. <br><br><br />
<br />
<span class="intro">For this purpose</span> three plasmids were constructed. The first expressing GFP from pTet promoter with no regulation. The second and third constitutively expressing TetR with or without the LVA tag as well<br><br><br />
<br />
<span class="intro">The pTet-GFP construct</span> was cloned.<br />
<br />
<span class="intro">A TetR (no LVA) construct</span> was cloned by PCR amplification without the LVA tag and addition of promoter, RBS and terminator. Subsequently the construct was ligated into the pTet-GFP construct. The constructs can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475004" target="_blank">Bba_K1475004</a> and <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>, respectively.<br><br><br />
<br />
<span class="intro">A TetR (with LVA) construct</span> was cloned by PCR amplification with the LVA tag and addition of the same promoter, RBS and terminator as TetR without LVA tag. Subsequently the construct was ligated into the pTet-GFP construct, as well. The construct can be found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in</span> the TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
<span class="intro">To test if the Tet promoter</span> could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
<span class = "intro">By using a strain,</span> constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
<span class = "intro">To analyse the amount</span> of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
<span class = "intro">Because pTet is leaky,</span> all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with doxycycline</span> were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<span class = "intro">Table 1:</span> Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
<br><br />
<span class="intro">Table 2:</span><br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP constructs</span> on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the growth</span> of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the growth</span> of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team proved</span> that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour40Team:SDU-Denmark/Tour402014-10-18T01:53:01Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Results</h3><br />
<br />
<p class='intro'><br />
<font color="3397FE">"I know that I am intelligent, because I know that I know nothing." – <b>Socrates</b></font><br />
</p><br />
<br />
<br />
<p><br />
<br />
<span class="intro">The next few pages will</span> guide you through the results of the characterization of our submitted parts. On this page, you will find short descriptions of our results, leading you to the final result. We hope you will dig deeper into the results of our systems. After the need to prioritize subprojects, the aim of our project was to get <i>E. coli</i> to express a self-designed nutritional protein, controlled by an inducible promoter. Furthermore it will express limonene synthase for the synthesis of limonene, the main part of lemon flavor.<br><br><br />
</p><br />
<br />
<h4>Synthesis of self-designed protein OneProt</h4><br />
<p><br />
Pictures of worms/WB<br><br />
Growth curve<br><br><br />
</p><br />
<br />
<h4>Controlling the Tet promoter</h4><br />
<p><br />
<a class="popupImg alignRight" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/4f/2014SDUresults5.png" title="Figure 2: 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)."><br />
<img src="https://static.igem.org/mediawiki/2014/1/19/2014SDUresults6.png" style="width:200px" /><br />
Figure 2: 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).<br />
</a><br />
<a class="popupImg alignRight" style="width:200px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b0/2014SDUresults3.png" title="Figure 1: 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)."><br />
<img src="https://static.igem.org/mediawiki/2014/e/e6/2014SDUresults4.png" style="width:200px" /><br />
Figure 1: 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).<br />
</a><br />
<br />
<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.<br />
</p><br />
<br><br><br><br />
<div><br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/12/2014SDUresults2.png" title="Figure 3: Dose response to doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/12/2014SDUresults2.png" style="width:500px" /><br />
Figure 3: Dose response to doxycycline.<br />
</a><br><br />
</div><br />
<br />
<h4>Flavor improvement</h4><br />
<p><br />
<span class="intro">The thought of eating <i>E. coli</i></span> does not sound that delicious – and that is why we want our OneProt to taste like lemon. Though the cloning proved unsuccessful. However, we have characterised an odor-free <i>E. coli</i> strain and compared it with a wild type K12 MG1655 <i>E. coli</i> strain. We have used Ion Mobility Spectroscopy to analyse the two strains, since we deemed it among the best methods to analyse odors. From the results it was shown that the odor-free strain does not produce indole, which is the compound responsible for the characteristic odor of <i>E. coli</i>. <br />
<br><br><br />
</p><br />
<br />
<h4>Added parts and devices</h4><br />
<p><br />
<span class="intro">To the great iGEM Registry of Standard Biological Parts</span> we have added 3 basic parts, 2 regulatory devices, 1 constitutively active production device and 4 regulable production devices.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour45Team:SDU-Denmark/Tour452014-10-18T01:45:12Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Submitted parts</h3><br />
<p><br />
<span class="intro">Below is a list</span> of the parts we submitted to the parts registry. To see sequencing, characterization etc. click the part names to go to the parts registry page.<br><br><br />
</p><br />
<p><br />
<br />
<br />
<div class="accordion" style="width:650px;"><br />
<br />
<div class="accordionTitel">Basic parts</div><br />
<div class="pane" ><br />
<br />
<br />
<span class="intro"><a href="http://parts.igem.org/Part:BBa_K1475001" target="_blank">BBa_K1475001</a></span><br><br />
<span class="intro">OneProt:</span><br />
The coding region of our self-designed nutritional protein. OneProt<br />
contains the recommended ratio of all the indispensible amino acids to a human <br />
diet. The protein is also compliant with the recommended ratio of indispensible <br />
to dispensable amino acids.<br />
<br />
<br><br><br />
<br />
<span class="intro"><a href="http://parts.igem.org/Part:BBa_K1475002" target="_blank">BBa_K1475002</a></span><br><br />
<span class="intro">fat-2:</span><br />
This part contains only the coding region of the fat-2 desaturase from<br />
<i>Caenorhabditis elegans</i>, the natural gene consists of three exons.<br />
<br />
<br><br><br />
<br />
<span class="intro"><a href="http://parts.igem.org/Part:BBa_K1475003" target="_blank">BBa_K1475003</a></span><br><br />
<span class="intro">TetR(N):</span><br />
the frequently used tet repressor from <i>Escherichia coli</i><br />
(<a href="http://parts.igem.org/Part:BBa_C0040">Part:BBa_C0040</a>) modified by removing the LVA rapid degradation tag.<br />
<br />
</div><br />
<br />
<div class="accordionTitel">Regulatory devices</div><br />
<div class="pane"><br />
<br />
<span class="intro"><a href="http://parts.igem.org/Part:BBa_K1475004">BBa_K1475004</a></span><br><br />
<span class="intro">Pcons-TetR(N)-term:</span><br />
the tet repressor from <i>Escherichia coli</i> is expressed<br />
form a constitutively active promoter. A terminator prevents transcription of <br />
genes downstream of the repressor.<br />
<br />
<br />
<br><br><br />
<br />
<br />
<span class="intro"><a href="http://parts.igem.org/Part:BBa_K1475008">BBa_K1475008</a></span><br><br />
<span class="intro">Pcon-LacI-term:</span><br />
Lac repressor protein derived from <i>Escherichia coli</i><br />
without LVA degradation tag, controlled by a constitutively active promoter.<br />
</div><br />
<br />
<div class="accordionTitel">Constitutively active production devices</div><br />
<div class="pane"><br />
<br />
<span class="intro"><a href="http://parts.igem.org/Part:BBa_K1475000">BBa_K1475000</a></span><br><br />
<span class="intro">p(tetR)-OneProt-term:</span><br />
OneProt is a self-designed protein designed to meet the<br />
recommended ratio of indispensible amino acids to a human diet according to <br />
the world health organization (WHO). It also contains the recommended ratio <br />
of indispensible to dispensible amino acids.<br />
<br />
<br />
<br />
</div><br />
<br />
<div class="accordionTitel">Regulable production devices</div><br />
<div class="pane current"><br />
<br />
<span class="intro"><a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a></span><br><br />
<span class="intro">Pcon-TetR(N)-term-p(TetR)-GFP-term:</span><br />
GFP controlled by a TetR, pTet expression system. The Tet repressor protein binds to the Tet promoter, the tet <br />
repressor protein is inhibited by aTc hereby inducing the expression of GFP.<br />
<br />
<br />
<br><br><br />
<br />
<br />
<span class="intro"><a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a></span><br><br />
<span class="intro">Pcon-TetR(LVA)-term-p(TetR)-GFP-term:</span><br />
GFP controlled by a TetR+LVA pTet<br />
expression system. The Tet repressor protein binds to the Tet promoter, the tet <br />
repressor protein is inhibited by aTc hereby inducing the expression of GFP.<br />
<br />
<br><br><br />
<br />
<br />
<span class="intro"><a href="http://parts.igem.org/Part:BBa_K1475007">BBa_K1475007</a></span><br><br />
<span class="intro">Pcon-LacI-term-p(LacI)-GFP-term:</span><br />
GFP controlled by the lac repressor and lac promoter system.<br />
<br />
<br><br><br />
<br />
<br />
<span class="intro"><a href="http://parts.igem.org/Part:BBa_K1475012">BBa_K1475012</a></span><br><br />
<span class="intro">Pcon-LacI-p(LacI)-GFP-term:</span><br />
GFP controlled by a LacI, p(LacI) system in which<br />
the LacI and p(Lac) sequence overlaps.<br />
</div><br />
</div><br />
</p><br />
<br><br><br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-18T00:06:06Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to investigate</span> the Tet promoter expression profile, and what influence the LVA tag had on this.<br><br><br />
<br />
<span class="intro">The pTet-GFP construct</span> was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us to ligate</span> the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
<span class="intro">The ligation of</span> TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in</span> the TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
<span class="intro">To test if the Tet promoter</span> could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
<span class = "intro">By using a strain,</span> constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
<span class = "intro">To analyse the amount</span> of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
<span class = "intro">Because pTet is leaky,</span> all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with doxycycline</span> were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<span class = "intro">Table 1:</span> Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
<br><br />
<span class="intro">Table 2:</span><br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP constructs</span> on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the growth</span> of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the growth</span> of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team proved</span> that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T23:57:38Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to investigate</span> the Tet promoter expression profile, and what influence the LVA tag had on this.<br><br><br />
<br />
<span class="intro">The ligation of</span> with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us to ligate</span> the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
<span class="intro">The ligation of</span> TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in</span> the TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
<span class="intro">To test if the Tet promoter</span> could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
<span class = "intro">By using a strain,</span> constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
<span class = "intro">To analyse the amount</span> of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
<span class = "intro">Because pTet is leaky,</span> all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with doxycycline</span> were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<span class = "intro">Table 1:</span> Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
<br><br />
<span class="intro">Table 2:</span><br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP constructs</span> on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the growth</span> of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the growth</span> of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team proved</span> that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T23:56:56Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to investigate</span> the Tet promoter expression profile, and what influence the LVA tag has on this.<br><br><br />
<br />
<span class="intro">The ligation of</span> with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us to ligate</span> the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
<span class="intro">The ligation of</span> TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in</span> the TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
<span class="intro">To test if the Tet promoter</span> could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
<span class = "intro">By using a strain,</span> constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
<span class = "intro">To analyse the amount</span> of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
<span class = "intro">Because pTet is leaky,</span> all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with doxycycline</span> were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<span class = "intro">Table 1:</span> Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
<br><br />
<span class="intro">Table 2:</span><br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP constructs</span> on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the growth</span> of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the growth</span> of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team proved</span> that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/File:2014SDUWestern_blot_with_GroL.jpgFile:2014SDUWestern blot with GroL.jpg2014-10-17T23:44:19Z<p>Danie12: uploaded a new version of &quot;File:2014SDUWestern blot with GroL.jpg&quot;: Figure 1: Western blot showing E. coli wild-type at OD600 at 0.3, 0.8 and 1.8 and E. coli expressing OneProt at OD600 at 0.3, 0.8, 1.8 and an overnight culture.</p>
<hr />
<div>Figure 1: Western blot showing <i>E. coli</i> wild-type at OD600 at 0.3, 0.8 and 1.8 and <i>E. coli</i> expressing OneProt at OD600 at 0.3, 0.8, 1.8 and an overnight culture.</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour42Team:SDU-Denmark/Tour422014-10-17T23:40:24Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>OneProt</h3><br />
<p><br />
<br />
<span class="intro">The pTet expression system</span> and limonene synthase construct is evolved around one thing: the OneProt.<br />
We have made the pTet-OneProt construct in order for us to synthesize a nutritional protein with the <br />
correct ratio of essential amino acids and the correct ratio between essential and non-essential <br />
amino acids. The device is found as <a href="http://parts.igem.org/Part:BBa_K1475000"> Bba_K1475000.</a><br><br><br />
<br />
<span class="intro">The protein is self-designed,</span> so we wanted to test if the protein were expressed in <i>E. coli</i> K12 MG1655, by<br />
the use of Western blotting. The western blot was blottet with <i>E. coli</i> K12 MG1655 wild-type and <i>E. coli</i><br />
expressing OneProt at different OD measures.<br><br><br />
</p><br />
<br />
<div class="popupImg alignCenter" style="width:500px"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4e/2014SDUWestern_blot_with_GroL.jpg" style="width:500px" /><br />
Figure 1: Western blot showing <i>E. coli</i> wild-type at OD600 at 0.3, 0.8 and 1.8 and <i>E. coli</i> expressing OneProt at OD600 at 0.3, 0.8, 1.8 and an overnight culture. <br />
</div><br />
<br />
<p><br />
<br><br />
<span class="intro">The protein has a</span> 3xFLAG tag and since bonds are showing, OneProt is expressed. However, from this<br />
western blot, we cannot see if the protein has been cut, just that it is expressed.<br />
</p><br />
<p><br />
<a class="popupImg alignRight" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" title="Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control."><br />
<img src="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" style="width:250px" /><br />
Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control.<br />
</a><br />
<br />
In order to check that we had the protein expressed in its full length, we did a coomassie stain on a SDS-<br />
page. Here we also wanted to receive information on the expression of the protein at different growth <br />
stages of <i>E. coli</i>. We analyzed samples from early exponential phase (OD600=0.3), late exponential phase <br />
(OD600=1.5), stationary phase (OD=2.5) and an overnight culture. As a control, <i>E. coli</i> with an empty vector <br />
(PSC1C3) was used.<br><br><br />
<br />
<span class="intro">OneProt has a molecular</span> weight of approximately 53.7 kDa. Unfortunately, there is no clear bond at this length. However, there is a bond at approximately 25 kDa, which is not detected in the control. We cannot <br />
conclude what gives rise to the band, but it might be a cellular response to an unfolded protein.<br><br><br />
<br />
<span class="intro">To test what effect</span> the expression of OneProt have on <i>E. coli</i> we set up a growth experiment<br />
measuring OD over time on the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 <br />
expressing OneProt and with an empty vector.<br><br><br />
<br />
</p><br />
<p><br />
<a class="popupImg alignLeft" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" title="Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector."><br />
<img src="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" style="width:250px" /><br />
Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector.<br />
</a><br />
<br />
From the growth curve, it is shown that the expression of OneProt stresses the metabolism a lot compared<br />
to the <i>E. coli</i> K12 wild-type. In addition to this, the metabolism of YYC912 is also quite stressed compared to <br />
the K12 wild-type. Despite the stressed metabolism of the two strains, the expression of OneProt increases <br />
over time as does the growth of YYC912.<br><br><br />
<br />
<span class="intro">By comparing the growth curve</span> of <i>E. coli</i> K12 expressing OneProt, TetR(+LVA), TetR(no LVA) and odor-free <i>E. coli</i> YYC912 it is seen that the metabolism of <i>E. coli</i> expressing OneProt and TetR is stressed compared to <br />
the wild-type, which means that it might be difficult to have OneProt expressed in high amounts controlled <br />
by pTet (+/-LVA). It is, however, shown that the cells are growing despite of their stressed metabolism <br />
and it is possible that the expression of OneProt can be controlled by pTet, which in turn is controlled by TetR, although <br />
the TetR(+LVA) seems more favorable. It can also be seen that the growth of <i>E. coli</i> YYC912 is comprised <br />
compared to the K12 wild-type which also contributes to possible difficulties in expressing OneProt in the <br />
odor-free YYC912 strain. However, the possibility still exists.<br><br><br />
</p><br />
<p><br />
<a class="popupImg alignRight" style="width:400px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" title="Figure 4: Growth curves showing <i>E. coli</i> K12 MG 1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:400px" /><br />
Figure 4: Growth curves showing <i>E. coli</i> K12 MG1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912.<br />
</a><br />
<br />
Because OneProt is self-designed, we wanted to test if the protein has any toxicity. To do so, we fed <br />
<i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector <br />
expressing OneProt on separate plates. On both plates, 20 <i>C. elegans</i> were tested. Articles recommend <br />
using heat shock assay for 7 hours: 1 hour at 35° C followed by 1 hour at 22°C, <br />
<span class="sourceReference">repeated.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Mosbech, M., et al.: Functional Loss of Two Ceramide Synthases Elicits Autophagy-Dependent Lifespan Extension in C. <br />
elegans.: PLoS ONE, 2013. 8 vol:7.<br />
<br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/23894595" target="_blank">(Link)</a></span><br />
<span class="sourceReference">&nbsp;</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Rodriguez, M., et al.:Worms under stress: <i>C. elegans</i> stress response and its relevance to complex human disease and <br />
aging. Trends in Genetics, 2013. Vol: 29, 6, p. 367-374.<br />
<br />
<a href="http://www.sciencedirect.com/science/article/pii/S016895251300022X" target="_blank">(Link)</a></span><br><br><br />
<br />
<span class="intro">After approximately 5 hours</span> no effects on <i>C. elegans</i> was detectable. Therefore we decided to stress <br />
<i>C. elegans</i> a little more, incubating them in 2 hours at 35°C followed by 1 hour at 22°C, repeated. After 7 <br />
hours, every <i>C. elegans</i> on both plates were alive. Thus we conclude that the protein has no toxic effect.<br><br><br />
</p><br />
<br />
<div class="popupImg alignCenter" style="width:400px" target="_blank" title="Figure 5:Picture of C. elegans fed with <i>E. coli</i> K12 MG1655 expressing OneProt."><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:400px" /><br />
Figure 5: Picture of C. elegans fed with <i>E. coli</i> K12 MG1655 expressing OneProt.<br />
</div><br />
<br><br><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/File:2014SDUWestern_blot_with_GroL.jpgFile:2014SDUWestern blot with GroL.jpg2014-10-17T23:39:40Z<p>Danie12: Figure 1: Western blot showing <i>E. coli</i> wild-type at OD600 at 0.3, 0.8 and 1.8 and <i>E. coli</i> expressing OneProt at OD600 at 0.3, 0.8, 1.8 and an overnight culture.</p>
<hr />
<div>Figure 1: Western blot showing <i>E. coli</i> wild-type at OD600 at 0.3, 0.8 and 1.8 and <i>E. coli</i> expressing OneProt at OD600 at 0.3, 0.8, 1.8 and an overnight culture.</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour42Team:SDU-Denmark/Tour422014-10-17T23:35:09Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>OneProt</h3><br />
<p><br />
<br />
<span class="intro">The pTet expression system</span> and limonene synthase construct is evolved around one thing: the OneProt.<br />
We have made the pTet-OneProt construct in order for us to synthesize a nutritional protein with the <br />
correct ratio of essential amino acids and the correct ratio between essential and non-essential <br />
amino acids. The device is found as <a href="http://parts.igem.org/Part:BBa_K1475000"> Bba_K1475000.</a><br><br><br />
<br />
<span class="intro">The protein is self-designed,</span> so we wanted to test if the protein were expressed in <i>E. coli</i> K12 MG1655, by<br />
the use of Western blotting. The western blot was blottet with <i>E. coli</i> K12 MG1655 wild-type and <i>E. coli</i><br />
expressing OneProt at different OD measures.<br><br><br />
</p><br />
<br />
<div class="popupImg alignCenter" style="width:500px"><br />
<img src="https://static.igem.org/mediawiki/2014/9/99/2014SDUoneprot5.png" style="width:500px" /><br />
Figure 1: Western blot showing <i>E. coli</i> wild-type and <i>E. coli</i> expressing OneProt at OD at 0.3, 0.8, 1.8 and an overnight <br />
culture. The membrane has been exposed for 10 minutes.<br />
</div><br />
<br />
<p><br />
<br><br />
<span class="intro">The protein has a</span> 3xFLAG tag and since bonds are showing, OneProt is expressed. However, from this<br />
western blot, we cannot see if the protein has been cut, just that it is expressed.<br />
</p><br />
<p><br />
<a class="popupImg alignRight" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" title="Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control."><br />
<img src="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" style="width:250px" /><br />
Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control.<br />
</a><br />
<br />
In order to check that we had the protein expressed in its full length, we did a coomassie stain on a SDS-<br />
page. Here we also wanted to receive information on the expression of the protein at different growth <br />
stages of <i>E. coli</i>. We analyzed samples from early exponential phase (OD600=0.3), late exponential phase <br />
(OD600=1.5), stationary phase (OD=2.5) and an overnight culture. As a control, <i>E. coli</i> with an empty vector <br />
(PSC1C3) was used.<br><br><br />
<br />
<span class="intro">OneProt has a molecular</span> weight of approximately 53.7 kDa. Unfortunately, there is no clear bond at this length. However, there is a bond at approximately 25 kDa, which is not detected in the control. We cannot <br />
conclude what gives rise to the band, but it might be a cellular response to an unfolded protein.<br><br><br />
<br />
<span class="intro">To test what effect</span> the expression of OneProt have on <i>E. coli</i> we set up a growth experiment<br />
measuring OD over time on the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 <br />
expressing OneProt and with an empty vector.<br><br><br />
<br />
</p><br />
<p><br />
<a class="popupImg alignLeft" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" title="Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector."><br />
<img src="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" style="width:250px" /><br />
Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector.<br />
</a><br />
<br />
From the growth curve, it is shown that the expression of OneProt stresses the metabolism a lot compared<br />
to the <i>E. coli</i> K12 wild-type. In addition to this, the metabolism of YYC912 is also quite stressed compared to <br />
the K12 wild-type. Despite the stressed metabolism of the two strains, the expression of OneProt increases <br />
over time as does the growth of YYC912.<br><br><br />
<br />
<span class="intro">By comparing the growth curve</span> of <i>E. coli</i> K12 expressing OneProt, TetR(+LVA), TetR(no LVA) and odor-free <i>E. coli</i> YYC912 it is seen that the metabolism of <i>E. coli</i> expressing OneProt and TetR is stressed compared to <br />
the wild-type, which means that it might be difficult to have OneProt expressed in high amounts controlled <br />
by pTet (+/-LVA). It is, however, shown that the cells are growing despite of their stressed metabolism <br />
and it is possible that the expression of OneProt can be controlled by pTet, which in turn is controlled by TetR, although <br />
the TetR(+LVA) seems more favorable. It can also be seen that the growth of <i>E. coli</i> YYC912 is comprised <br />
compared to the K12 wild-type which also contributes to possible difficulties in expressing OneProt in the <br />
odor-free YYC912 strain. However, the possibility still exists.<br><br><br />
</p><br />
<p><br />
<a class="popupImg alignRight" style="width:400px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" title="Figure 4: Growth curves showing <i>E. coli</i> K12 MG 1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:400px" /><br />
Figure 4: Growth curves showing <i>E. coli</i> K12 MG1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912.<br />
</a><br />
<br />
Because OneProt is self-designed, we wanted to test if the protein has any toxicity. To do so, we fed <br />
<i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector <br />
expressing OneProt on separate plates. On both plates, 20 <i>C. elegans</i> were tested. Articles recommend <br />
using heat shock assay for 7 hours: 1 hour at 35° C followed by 1 hour at 22°C, <br />
<span class="sourceReference">repeated.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Mosbech, M., et al.: Functional Loss of Two Ceramide Synthases Elicits Autophagy-Dependent Lifespan Extension in C. <br />
elegans.: PLoS ONE, 2013. 8 vol:7.<br />
<br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/23894595" target="_blank">(Link)</a></span><br />
<span class="sourceReference">&nbsp;</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Rodriguez, M., et al.:Worms under stress: <i>C. elegans</i> stress response and its relevance to complex human disease and <br />
aging. Trends in Genetics, 2013. Vol: 29, 6, p. 367-374.<br />
<br />
<a href="http://www.sciencedirect.com/science/article/pii/S016895251300022X" target="_blank">(Link)</a></span><br><br><br />
<br />
<span class="intro">After approximately 5 hours</span> no effects on <i>C. elegans</i> was detectable. Therefore we decided to stress <br />
<i>C. elegans</i> a little more, incubating them in 2 hours at 35°C followed by 1 hour at 22°C, repeated. After 7 <br />
hours, every <i>C. elegans</i> on both plates were alive. Thus we conclude that the protein has no toxic effect.<br><br><br />
</p><br />
<br />
<div class="popupImg alignCenter" style="width:400px" target="_blank" title="Figure 5:Picture of C. elegans fed with <i>E. coli</i> K12 MG1655 expressing OneProt."><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:400px" /><br />
Figure 5: Picture of C. elegans fed with <i>E. coli</i> K12 MG1655 expressing OneProt.<br />
</div><br />
<br><br><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/File:2014SDUGrowth_-_WT,_YYC912,_Oneprot,_Empty_vector.pngFile:2014SDUGrowth - WT, YYC912, Oneprot, Empty vector.png2014-10-17T23:34:14Z<p>Danie12: Figure 3: Growth curve illustrating the growth of E. coli K12 MG1655 WT, odor-free E. coli YYC912, E. coli K12 expressing OneProt and with an empty vector.</p>
<hr />
<div>Figure 3: Growth curve illustrating the growth of E. coli K12 MG1655 WT, odor-free E. coli YYC912, E. coli K12 expressing OneProt and with an empty vector.</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour42Team:SDU-Denmark/Tour422014-10-17T23:33:04Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>OneProt</h3><br />
<p><br />
<br />
<span class="intro">The pTet expression system</span> and limonene synthase construct is evolved around one thing: the OneProt.<br />
We have made the pTet-OneProt construct in order for us to synthesize a nutritional protein with the <br />
correct ratio of essential amino acids and the correct ratio between essential and non-essential <br />
amino acids. The device is found as <a href="http://parts.igem.org/Part:BBa_K1475000"> Bba_K1475000.</a><br><br><br />
<br />
<span class="intro">The protein is self-designed,</span> so we wanted to test if the protein were expressed in <i>E. coli</i> K12 MG1655, by<br />
the use of Western blotting. The western blot was blottet with <i>E. coli</i> K12 MG1655 wild-type and <i>E. coli</i><br />
expressing OneProt at different OD measures.<br><br><br />
</p><br />
<br />
<div class="popupImg alignCenter" style="width:500px"><br />
<img src="https://static.igem.org/mediawiki/2014/9/99/2014SDUoneprot5.png" style="width:500px" /><br />
Figure 1: Western blot showing <i>E. coli</i> wild-type and <i>E. coli</i> expressing OneProt at OD at 0.3, 0.8, 1.8 and an overnight <br />
culture. The membrane has been exposed for 10 minutes.<br />
</div><br />
<br />
<p><br />
<br><br />
<span class="intro">The protein has a</span> 3xFLAG tag and since bonds are showing, OneProt is expressed. However, from this<br />
western blot, we cannot see if the protein has been cut, just that it is expressed.<br />
</p><br />
<p><br />
<a class="popupImg alignRight" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" title="Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control."><br />
<img src="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" style="width:250px" /><br />
Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control.<br />
</a><br />
<br />
In order to check that we had the protein expressed in its full length, we did a coomassie stain on a SDS-<br />
page. Here we also wanted to receive information on the expression of the protein at different growth <br />
stages of <i>E. coli</i>. We analyzed samples from early exponential phase (OD600=0.3), late exponential phase <br />
(OD600=1.5), stationary phase (OD=2.5) and an overnight culture. As a control, <i>E. coli</i> with an empty vector <br />
(PSC1C3) was used.<br><br><br />
<br />
<span class="intro">OneProt has a molecular</span> weight of approximately 53.7 kDa. Unfortunately, there is no clear bond at this length. However, there is a bond at approximately 25 kDa, which is not detected in the control. We cannot <br />
conclude what gives rise to the band, but it might be a cellular response to an unfolded protein.<br><br><br />
<br />
<span class="intro">To test what effect</span> the expression of OneProt have on <i>E. coli</i> we set up a growth experiment<br />
measuring OD over time on the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 <br />
expressing OneProt and with an empty vector.<br><br><br />
<br />
</p><br />
<p><br />
<a class="popupImg alignLeft" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/e/e5/2014SDUresults8.png" title="Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector."><br />
<img src="https://static.igem.org/mediawiki/2014/e/e5/2014SDUresults8.png" style="width:250px" /><br />
Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector.<br />
</a><br />
<br />
From the growth curve, it is shown that the expression of OneProt stresses the metabolism a lot compared<br />
to the <i>E. coli</i> K12 wild-type. In addition to this, the metabolism of YYC912 is also quite stressed compared to <br />
the K12 wild-type. Despite the stressed metabolism of the two strains, the expression of OneProt increases <br />
over time as does the growth of YYC912.<br><br><br />
<br />
<span class="intro">By comparing the growth curve</span> of <i>E. coli</i> K12 expressing OneProt, TetR(+LVA), TetR(no LVA) and odor-free <i>E. coli</i> YYC912 it is seen that the metabolism of <i>E. coli</i> expressing OneProt and TetR is stressed compared to <br />
the wild-type, which means that it might be difficult to have OneProt expressed in high amounts controlled <br />
by pTet (+/-LVA). It is, however, shown that the cells are growing despite of their stressed metabolism <br />
and it is possible that the expression of OneProt can be controlled by pTet, which in turn is controlled by TetR, although <br />
the TetR(+LVA) seems more favorable. It can also be seen that the growth of <i>E. coli</i> YYC912 is comprised <br />
compared to the K12 wild-type which also contributes to possible difficulties in expressing OneProt in the <br />
odor-free YYC912 strain. However, the possibility still exists.<br><br><br />
</p><br />
<p><br />
<a class="popupImg alignRight" style="width:400px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" title="Figure 4: Growth curves showing <i>E. coli</i> K12 MG 1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:400px" /><br />
Figure 4: Growth curves showing <i>E. coli</i> K12 MG1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912.<br />
</a><br />
<br />
Because OneProt is self-designed, we wanted to test if the protein has any toxicity. To do so, we fed <br />
<i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector <br />
expressing OneProt on separate plates. On both plates, 20 <i>C. elegans</i> were tested. Articles recommend <br />
using heat shock assay for 7 hours: 1 hour at 35° C followed by 1 hour at 22°C, <br />
<span class="sourceReference">repeated.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Mosbech, M., et al.: Functional Loss of Two Ceramide Synthases Elicits Autophagy-Dependent Lifespan Extension in C. <br />
elegans.: PLoS ONE, 2013. 8 vol:7.<br />
<br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/23894595" target="_blank">(Link)</a></span><br />
<span class="sourceReference">&nbsp;</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Rodriguez, M., et al.:Worms under stress: <i>C. elegans</i> stress response and its relevance to complex human disease and <br />
aging. Trends in Genetics, 2013. Vol: 29, 6, p. 367-374.<br />
<br />
<a href="http://www.sciencedirect.com/science/article/pii/S016895251300022X" target="_blank">(Link)</a></span><br><br><br />
<br />
<span class="intro">After approximately 5 hours</span> no effects on <i>C. elegans</i> was detectable. Therefore we decided to stress <br />
<i>C. elegans</i> a little more, incubating them in 2 hours at 35°C followed by 1 hour at 22°C, repeated. After 7 <br />
hours, every <i>C. elegans</i> on both plates were alive. Thus we conclude that the protein has no toxic effect.<br><br><br />
</p><br />
<br />
<div class="popupImg alignCenter" style="width:400px" target="_blank" title="Figure 5:Picture of C. elegans fed with <i>E. coli</i> K12 MG1655 expressing OneProt."><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/2014SDUresults10.jpg" style="width:400px" /><br />
Figure 5: Picture of C. elegans fed with <i>E. coli</i> K12 MG1655 expressing OneProt.<br />
</div><br />
<br><br><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T23:23:21Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to test</span> if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on <br />
the expression.<br><br><br />
<br />
The ligation of the TetR (+LVA) construct with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us</span> to ligate the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
The ligation of TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in the</span> TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
By using a strain constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with</span> doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
Table 1: Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
Table 2:<br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP</span> constructs on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the</span> growth of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the</span> growth of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team</span> proved that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T23:20:19Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to test</span> if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on <br />
the expression.<br><br><br />
<br />
The ligation of the TetR (+LVA) construct with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us</span> to ligate the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
The ligation of TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in the</span> TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
By using a strain constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with</span> doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
Table 1: Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
<br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP</span> constructs on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the</span> growth of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the</span> growth of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team</span> proved that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T23:19:17Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to test</span> if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on <br />
the expression.<br><br><br />
<br />
The ligation of the TetR (+LVA) construct with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us</span> to ligate the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
The ligation of TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in the</span> TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
By using a strain constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with</span> doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
Table 1: Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
</table><br />
<br><br><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
Table 2:<br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP</span> constructs on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the</span> growth of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the</span> growth of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team</span> proved that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/File:2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNGFile:2014SDUDouble growth curve - revideret daniel-01-01.PNG2014-10-17T23:18:33Z<p>Danie12: Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.</p>
<hr />
<div>Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T23:13:39Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to test</span> if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on <br />
the expression.<br><br><br />
<br />
The ligation of the TetR (+LVA) construct with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us</span> to ligate the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
The ligation of TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in the</span> TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
By using a strain constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with</span> doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
Table 1: Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
</table><br />
<br><br><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
Table 2:<br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP</span> constructs on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the</span> growth of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/0/08/2014SDUDouble_growth_curve_-_revideret_daniel-01.PNG" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the</span> growth of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team</span> proved that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/File:2014SDUDouble_growth_curve_-_revideret_daniel-01.PNGFile:2014SDUDouble growth curve - revideret daniel-01.PNG2014-10-17T23:13:14Z<p>Danie12: Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.</p>
<hr />
<div>Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T23:07:59Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to test</span> if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on <br />
the expression.<br><br><br />
<br />
The ligation of the TetR (+LVA) construct with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us</span> to ligate the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
The ligation of TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in the</span> TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
By using a strain constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with</span> doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
Table 1: Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
</table><br />
<br><br><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
Table 2:<br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP</span> constructs on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the</span> growth of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e3/2014SDUexpressions31.png" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the</span> growth of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team</span> proved that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/File:SDU2014expressionsCoomassie_TetR_rettet_2000.pngFile:SDU2014expressionsCoomassie TetR rettet 2000.png2014-10-17T23:06:22Z<p>Danie12: Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.</p>
<hr />
<div>Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T23:01:48Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to test</span> if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on <br />
the expression.<br><br><br />
<br />
The ligation of the TetR (+LVA) construct with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us</span> to ligate the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
The ligation of TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in the</span> TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
By using a strain constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/23/2014SDUexpressions32.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/2/23/2014SDUexpressions32.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with</span> doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
Table 1: Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
</table><br />
<br><br><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
Table 2:<br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP</span> constructs on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the</span> growth of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e3/2014SDUexpressions31.png" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the</span> growth of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team</span> proved that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T22:59:14Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to test</span> if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on <br />
the expression.<br><br><br />
<br />
The ligation of the TetR (+LVA) construct with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us</span> to ligate the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
The ligation of TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in the</span> TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
By using a strain constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/23/2014SDUexpressions32.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/2/23/2014SDUexpressions32.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with</span> doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
Table 1: Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
</table><br />
<br><br><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
Table 2:<br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP</span> constructs on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the</span> growth of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e3/2014SDUexpressions31.png" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the</span> growth of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team</span> proved that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T22:58:36Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to test</span> if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on <br />
the expression.<br><br><br />
<br />
The ligation of the TetR (+LVA) construct with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us</span> to ligate the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
The ligation of TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in the</span> TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
By using a strain constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/23/2014SDUexpressions32.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/2/23/2014SDUexpressions32.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with</span> doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
Table 1: Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the dublicates was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.<br />
</div><br />
<div><br />
</table><br />
<br><br><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
Table 2:<br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP</span> constructs on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the</span> growth of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e3/2014SDUexpressions31.png" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the</span> growth of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team</span> proved that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T22:57:27Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to test</span> if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on <br />
the expression.<br><br><br />
<br />
The ligation of the TetR (+LVA) construct with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us</span> to ligate the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
The ligation of TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in the</span> TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
By using a strain constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/23/2014SDUexpressions32.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/2/23/2014SDUexpressions32.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with</span> doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<div><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
Table 1: Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the dublicates was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">Bba_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">Bba_K1475006</a>.<br />
</div><br />
<div><br />
</table><br />
<br><br><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</div><br />
Table 2:<br />
</table><br />
<br><br />
<p><br />
<span class="intro">The plating of TetR-GFP</span> constructs on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the</span> growth of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e3/2014SDUexpressions31.png" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the</span> growth of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team</span> proved that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T22:29:40Z<p>Danie12: </p>
<hr />
<div>{{:Team:SDU-Denmark/core/header| }}<br />
<br />
<html><br />
<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to test</span> if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on <br />
the expression.<br><br><br />
<br />
The ligation of the TetR (+LVA) construct with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us</span> to ligate the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
The ligation of TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in the</span> TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
By using a strain constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/23/2014SDUexpressions32.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/2/23/2014SDUexpressions32.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with</span> doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</table><br />
<br><br><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</table><br><br />
<p><br />
<span class="intro">The plating of TetR-GFP</span> constructs on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the</span> growth of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e3/2014SDUexpressions31.png" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the</span> growth of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team</span> proved that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
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{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour41Team:SDU-Denmark/Tour412014-10-17T22:21:35Z<p>Danie12: </p>
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<h3>Expressions</h3><br />
<h4>Characerization of TetR/pTet</h4><br />
<p><br />
<span class="intro">We wanted to test</span> if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on <br />
the expression.<br><br><br />
<br />
The ligation of the TetR (+LVA) construct with the pTet-GFP construct was cloned and is found as <br />
<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br><br />
<br />
<span class="intro">In order for us</span> to ligate the TetR (no LVA) construct with the pTet-GFP construct, we first needed<br />
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be <br />
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br><br />
<br />
The ligation of TetR (no LVA) construct with the pTet-GFP construct was cloned and can<br />
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br><br />
</p><br />
<br />
<h4>Characteriztion/expression</h4><br />
<p><br />
<span class="intro">The promoters in the</span> TetR-pTet constructs are supposed to be inhibited by TetR. By induction with<br />
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed <br />
after induction with <br />
<span class="sourceReference">doxycycline.</span><br />
<span class="tooltip"><br />
<span class="tooltipHeader">Source:</span><br />
Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression<br />
of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular <br />
Therapy, 2007. 15:5, p. 938-945.<br />
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br><br />
To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS<br />
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with <br />
and without LVA tag. A wild-type was used as control.<br><br><br />
</p><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter."><br />
<img src="https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png" style="width:500px" /><br />
Figure 1: Results of the FACS before and after induction with doxycycline.<br />
</a><br />
<p><br />
<br><br />
<span class="intro">The results of the FACS</span> illustrates that without induction with doxycycline, GFP is still expressed. This is because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is makedly reduces in the constructs containing tetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br><br />
<br />
<span class="intro">Leaving the leakiness of pTet</span> out of account, FACS results indicates that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins, this can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.<br><br><br />
<br />
By using a strain constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer. <br />
<br><br><br />
<br />
To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was<br />
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, <br />
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR <br />
(+LVA)-GFP, respectively.<br><br><br />
<br />
<a class="popupImg alignCenter" style="width:500px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/23/2014SDUexpressions32.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively."><br />
<img src="https://static.igem.org/mediawiki/2014/2/23/2014SDUexpressions32.png" style="width:500px" /><br />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.<br />
</a><br />
<br><br />
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the<br />
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) <br />
in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) <br />
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the <br />
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due <br />
to the LVA tag making TetR unstable and tagging it for degredation. <br><br><br />
<br />
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to <br />
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS <br />
clearly shows an induction.<br><br><br />
<br />
<span class="intro">Duplicates of plates with</span> doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 <br />
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet <br />
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br><br />
</p><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">First series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/2/2d/2014SDUexpressions5.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/2/2f/2014SDUexpressions2.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5d/2014SDUexpressions6.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUexpressions3.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions7.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/a/a1/2014SDUexpressions4.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/d/d0/2014SDUexpressions12.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1a/2014SDUexpressions8.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions13.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/17/2014SDUexpressions9.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/3/37/2014SDUexpressions14.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/57/2014SDUexpressions10.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/c3/2014SDUexpressions15.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/5/56/2014SDUexpressions11.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</table><br />
<br><br><br />
<table frame="box" rules="rows" style="width:800px"><br />
<tr><br />
<td style="width:200px"></td><br />
<td style="width:200px"> 0 ng/mL doxycycline</td><br />
<td style="width:200px"> 50 ng/mL doxycycline</td><br />
<td style="width:200px"> 100 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/41/2014SDUexpressions16.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions17.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/d/d3/2014SDUexpressions18.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
<tr><br />
<td style="width:200px">200 ng/mL doxycycline</td><br />
<td style="width:200px">500 ng/mL doxycycline</td><br />
<td style="width:200px">1000 ng/mL doxycycline</td><br />
<td style="width:200px">2000 ng/mL doxycycline</td><br />
</tr><br />
<tr><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/2014SDUexpressions19.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/70/2014SDUexpressions20.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/1/1e/2014SDUexpressions21.png" style="width:180px" /><br />
</a><br />
</td><br />
<td style="width:200px"> <br />
<a class="popupImg alignCenter" style="width:180px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline."><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/2014SDUexpressions22.png" style="width:180px" /><br />
</a><br />
</td><br />
</tr><br />
</table><br><br />
<p><br />
<span class="intro">The plating of</span> TetR-GFP constructs on plates with doxycycline shows that GFP is expressed at different <br />
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline <br />
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This <br />
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS <br />
results.<br><br><br />
<br />
<span class="intro">To see how the</span> growth of the bacteria expressing GFP controlled by pTet are affected, we have measured <br />
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-<br />
GFP, pTet (+LVA)-GFP and a wild-type.<br><br><br />
</p><br />
<div class="popupImg alignCenter" style="width:800px"><br />
<img src="https://static.igem.org/mediawiki/2014/e/e3/2014SDUexpressions31.png" style="width:800px" /><br />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.<br />
</div><br />
<p><br />
<br><br />
<span class="intro">Figure 3 shows the</span> growth of bacteria expressing GFP constitutiely, are attenuated the most with most <br />
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the <br />
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR<br />
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR <br />
without LVA.</p><br><br><br />
<br />
<h4>Characterization of lacI/plac</h4><br />
<p><br />
<span class="intro">2013 SDU-Denmark iGEM team</span> proved that the natural lac inhibitor has a faster respondance on induction <br />
by IPTG, than lacI with LVA (<a href="https://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted <br />
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without <br />
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully <br />
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.<br />
<br><br><br><br />
</p><br />
<br />
</html><br />
<br />
{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/Team:SDU-Denmark/Tour60Team:SDU-Denmark/Tour602014-10-15T17:17:19Z<p>Danie12: </p>
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{{:Team:SDU-Denmark/core/footer}}</div>Danie12http://2014.igem.org/File:2014SDUScene_4_2_-_Medium.movFile:2014SDUScene 4 2 - Medium.mov2014-10-15T12:26:37Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_4_1_-_Medium.movFile:2014SDUScene 4 1 - Medium.mov2014-10-15T12:24:30Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_3_2_%2B_4_-_Medium.movFile:2014SDUScene 3 2 + 4 - Medium.mov2014-10-15T12:22:18Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_3_1_%2B_4_-_Medium.movFile:2014SDUScene 3 1 + 4 - Medium.mov2014-10-15T12:21:31Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_2_2_%2B_3_-_Medium.movFile:2014SDUScene 2 2 + 3 - Medium.mov2014-10-15T12:20:55Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_2_1_%2B_3_-_Medium.movFile:2014SDUScene 2 1 + 3 - Medium.mov2014-10-15T12:19:50Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_1_2_3_%2B_1_1_1_-_Medium.movFile:2014SDUScene 1 2 3 + 1 1 1 - Medium.mov2014-10-15T12:19:07Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_1_2_2_%2B_1_1_1_-_Medium.movFile:2014SDUScene 1 2 2 + 1 1 1 - Medium.mov2014-10-15T12:18:32Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_1_2_%2B_1_2_1_OK_-_Medium.movFile:2014SDUScene 1 2 + 1 2 1 OK - Medium.mov2014-10-15T12:17:54Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_1_1_3_4_%2B_1_1_3_5_%2B_2_-_Medium.movFile:2014SDUScene 1 1 3 4 + 1 1 3 5 + 2 - Medium.mov2014-10-15T12:17:21Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_1_1_3_3_%2B_1_1_3_5_%2B_2_-_Medium.movFile:2014SDUScene 1 1 3 3 + 1 1 3 5 + 2 - Medium.mov2014-10-15T12:16:41Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_1_1_3_2_%2B_1_1_3_5_%2B_2_-_Medium.movFile:2014SDUScene 1 1 3 2 + 1 1 3 5 + 2 - Medium.mov2014-10-15T12:15:50Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_1_1_3_1_%2B_1_1_3_5_%2B_2_-_Medium.movFile:2014SDUScene 1 1 3 1 + 1 1 3 5 + 2 - Medium.mov2014-10-15T12:14:58Z<p>Danie12: </p>
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<div></div>Danie12http://2014.igem.org/File:2014SDUScene_1_1_3_-_Medium.movFile:2014SDUScene 1 1 3 - Medium.mov2014-10-15T12:13:54Z<p>Danie12: </p>
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<div></div>Danie12