http://2013.igem.org/wiki/index.php?title=Team:DTU-Denmark/HelloWorld&feed=atom&action=historyTeam:DTU-Denmark/HelloWorld - Revision history2024-03-30T03:26:25ZRevision history for this page on the wikiMediaWiki 1.16.5http://2013.igem.org/wiki/index.php?title=Team:DTU-Denmark/HelloWorld&diff=298969&oldid=prevHelencook: /* Conclusions */2013-10-04T18:01:27Z<p><span class="autocomment">Conclusions</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Conclusions ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Conclusions ==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Biobrick [[Team:DTU-Denmark/Parts|BBa_K1067009]] successfully directs GFP SF to the periplasm of ''E. coli''. This experiment is a proof of concept that proteins can be exported to the periplasm. <del class="diffchange diffchange-inline">There have in </del>previous iGEM <del class="diffchange diffchange-inline">project </del>been <del class="diffchange diffchange-inline">a confusing </del>whether or not it <del class="diffchange diffchange-inline">was </del>possible to export GFP to the periplasm; this experiment verifies just that and gives a procedure on how to reproduce this. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Biobrick [[Team:DTU-Denmark/Parts|BBa_K1067009]] successfully directs GFP SF to the periplasm of ''E. coli''. This experiment is a proof of concept that proteins can be exported to the periplasm. <ins class="diffchange diffchange-inline">In </ins>previous iGEM <ins class="diffchange diffchange-inline">projects, there has </ins>been <ins class="diffchange diffchange-inline">confusion as to </ins>whether or not it <ins class="diffchange diffchange-inline">is </ins>possible to export GFP to the periplasm; this experiment verifies just that and gives a procedure on how to reproduce this.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==References== </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==References== </div></td></tr>
</table>Helencookhttp://2013.igem.org/wiki/index.php?title=Team:DTU-Denmark/HelloWorld&diff=298936&oldid=prevHelencook: /* Periplasmic vs. cytoplasmic fraction */2013-10-04T18:00:15Z<p><span class="autocomment">Periplasmic vs. cytoplasmic fraction</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Periplasmic vs. cytoplasmic fraction===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Periplasmic vs. cytoplasmic fraction===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The first test we did was to purify the periplasmic fraction of the cells and then <del class="diffchange diffchange-inline">comparing </del>with the cytoplasmic ([https://2013.igem.org/Team:DTU-Denmark/Notebook/6_July_2013 <del class="diffchange diffchange-inline">link to </del>notebook]). We did this with the cold osmotic shock method and viewed the fraction under UV-light. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The first test we did was to purify the periplasmic fraction of the cells and then <ins class="diffchange diffchange-inline">to compare them </ins>with the cytoplasmic <ins class="diffchange diffchange-inline">fraction </ins>([https://2013.igem.org/Team:DTU-Denmark/Notebook/6_July_2013 <ins class="diffchange diffchange-inline">lab </ins>notebook <ins class="diffchange diffchange-inline">entry</ins>]). We did this with the cold osmotic shock method and viewed the fraction under UV-light. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:DTU-Denmark_HelloWorld_Sucrose_A+D+C_2.jpg|thumb|center|upright=3|Periplasmic fractions are clear green fluorescent while the pellet with the cytoplasmic fraction is red fluorescent]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:DTU-Denmark_HelloWorld_Sucrose_A+D+C_2.jpg|thumb|center|upright=3|Periplasmic fractions are clear green fluorescent while the pellet with the cytoplasmic fraction is red fluorescent]]</div></td></tr>
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</table>Helencookhttp://2013.igem.org/wiki/index.php?title=Team:DTU-Denmark/HelloWorld&diff=298916&oldid=prevHelencook: /* Visualization */2013-10-04T17:59:14Z<p><span class="autocomment">Visualization</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Visualization===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Visualization===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To visualize that GFP SF actually is exported we constructed a procedure for growing the cells and tracing the GFP SF to the periplasm (Skoog, Karl, et al.). <del class="diffchange diffchange-inline">It turns out that </del>if you just grow cells in LB and induce the GFP SF/RFP expression there will be too much GFP SF in the cytoplasm to get a good resolution between cytoplasm and periplasm. That is why it’s important to devise a procedure for stopping the production for GFP SP and thereafter tracing it from the cytoplasm into the periplasm. This underlines the importance of having a non-leaky inducible expression system; when expression is stopped by removing the inducer it would spoil the procedure if the system kept leaking out GFP SF into the cytoplasm.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To visualize that GFP SF actually is exported<ins class="diffchange diffchange-inline">, </ins>we constructed a procedure for growing the cells and tracing the GFP SF to the periplasm (Skoog, Karl, et al.). <ins class="diffchange diffchange-inline">As we initially found, </ins>if you just grow cells in LB and induce the GFP SF/RFP expression<ins class="diffchange diffchange-inline">, </ins>there will be too much GFP SF in the cytoplasm to get a good resolution between cytoplasm and periplasm. That is why it’s important to devise a procedure for stopping the production for GFP SP and thereafter tracing it from the cytoplasm into the periplasm. This underlines the importance of having a non-leaky inducible expression system; when expression is stopped by removing the inducer it would spoil the procedure if the system kept leaking out GFP SF into the cytoplasm.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After optimizing the procedure we made a final version which can be found in the [https://2013.igem.org/Team:DTU-Denmark/Methods/Visualizing_GFP_in_the_periplasm methods page]. This also includes how to use background subtraction to get a better resolution.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>After optimizing the procedure we made a final version which can be found in the [https://2013.igem.org/Team:DTU-Denmark/Methods/Visualizing_GFP_in_the_periplasm methods page]. This also includes how to use background subtraction to get a better resolution.</div></td></tr>
</table>Helencookhttp://2013.igem.org/wiki/index.php?title=Team:DTU-Denmark/HelloWorld&diff=298893&oldid=prevHelencook: /* Periplasmic vs. cytoplasmic fraction */2013-10-04T17:58:03Z<p><span class="autocomment">Periplasmic vs. cytoplasmic fraction</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Periplasmic vs. cytoplasmic fraction===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Periplasmic vs. cytoplasmic fraction===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The first test we did was to purify the periplasmic fraction of the cells and then comparing with the cytoplasmic ([https://2013.igem.org/Team:DTU-Denmark/Notebook/6_July_2013 link to notebook]). We did this with the cold osmotic shock method and viewed the fraction under UV-light. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The first test we did was to purify the periplasmic fraction of the cells and then comparing with the cytoplasmic ([https://2013.igem.org/Team:DTU-Denmark/Notebook/6_July_2013 link to notebook]). We did this with the cold osmotic shock method and viewed the fraction under UV-light. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[File:<del class="diffchange diffchange-inline">Sucrose A</del>+D+<del class="diffchange diffchange-inline">C</del>.<del class="diffchange diffchange-inline">JPG</del>|thumb|center|upright=3|Periplasmic fractions are clear green fluorescent while the pellet with the cytoplasmic fraction is red fluorescent]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[File:<ins class="diffchange diffchange-inline">DTU-Denmark_HelloWorld_Sucrose_A</ins>+D+<ins class="diffchange diffchange-inline">C_2</ins>.<ins class="diffchange diffchange-inline">jpg</ins>|thumb|center|upright=3|Periplasmic fractions are clear green fluorescent while the pellet with the cytoplasmic fraction is red fluorescent]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Fluorescent microscope images=== </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Fluorescent microscope images=== </div></td></tr>
</table>Helencookhttp://2013.igem.org/wiki/index.php?title=Team:DTU-Denmark/HelloWorld&diff=298851&oldid=prevHelencook: /* Construction */2013-10-04T17:56:20Z<p><span class="autocomment">Construction</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The TAT signal peptide was bought as a gBlock from IDT. All fragments were assembled into an expression vector specially designed to have a tight on/off mechanism. For this we used a promoter constructed from our [[Team:DTU-Denmark/pBAD_SPL synthetic promoter library]] ([http://parts.igem.org/Part:BBa_K1067007 BBa_K1067007]). Primers were design by the program [http://www.cbs.dtu.dk/services/PHUSER-2.0/web/ PHUSER] [5] so that we got a seamless assembly.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The TAT signal peptide was bought as a gBlock from IDT. All fragments were assembled into an expression vector specially designed to have a tight on/off mechanism. For this we used a promoter constructed from our [[Team:DTU-Denmark/pBAD_SPL<ins class="diffchange diffchange-inline">|</ins>synthetic promoter library]] ([http://parts.igem.org/Part:BBa_K1067007 BBa_K1067007]). Primers were design by the program [http://www.cbs.dtu.dk/services/PHUSER-2.0/web/ PHUSER] [5] so that we got a seamless assembly.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:PZA21-MCS TAT araBAD.png|680px|alt=Alt|"Hello World"" construct]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:PZA21-MCS TAT araBAD.png|680px|alt=Alt|"Hello World"" construct]]</div></td></tr>
</table>Helencookhttp://2013.igem.org/wiki/index.php?title=Team:DTU-Denmark/HelloWorld&diff=298843&oldid=prevHelencook: /* Construction */2013-10-04T17:56:06Z<p><span class="autocomment">Construction</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Methods ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Methods ==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Construction===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Construction===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The overal goal was to test the twin arginine pathway (TAT) and whether this signal peptide could transport GFP SF into the periplasm. The reason why we choose to use GFP superfolder (SF) was that this variant has been shown to fold faster than the E. coli transport system is at translocation, [3]. This assures that the GFP will form its fluorophore before translocation to the more reductive periplasmic space. Thus it will not be inactivated by the inhibition of fluorophore formation as seen in other oxidative compartments like ER [4].</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The overal goal was to test the twin arginine pathway (TAT) and whether this signal peptide could transport GFP SF into the periplasm. The reason why we choose to use GFP superfolder (SF) was that this variant has been shown to fold faster than the <ins class="diffchange diffchange-inline">''</ins>E. coli<ins class="diffchange diffchange-inline">'' </ins>transport system is at translocation, [3]. This assures that the GFP will form its fluorophore before translocation to the more reductive periplasmic space. Thus it will not be inactivated by the inhibition of fluorophore formation as seen in other oxidative compartments like <ins class="diffchange diffchange-inline">the </ins>ER [4].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Construction of the plasmids was done with USER-cloning and assembly of 3 fragments. The starting point was a plasmid construct with RFP and GFP SF respectively. The RFP and GFP SF were amplified out with their associated RBS (the same in both cases). </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Construction of the plasmids was done with USER-cloning and assembly of 3 fragments. The starting point was a plasmid construct with RFP and GFP SF respectively. The RFP and GFP SF were amplified out with their associated RBS (the same in both cases). </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The TAT signal peptide was bought as a gBlock from IDT. All fragments were assembled into an expression vector specially designed to have a tight on/off mechanism ([http://parts.igem.org/Part:BBa_K1067007 BBa_K1067007]). Primers were design by the program [http://www.cbs.dtu.dk/services/PHUSER-2.0/web/ PHUSER] [5] so that we got a seamless assembly.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The TAT signal peptide was bought as a gBlock from IDT. All fragments were assembled into an expression vector specially designed to have a tight on/off mechanism<ins class="diffchange diffchange-inline">. For this we used a promoter constructed from our [[Team:DTU-Denmark/pBAD_SPL synthetic promoter library]] </ins>([http://parts.igem.org/Part:BBa_K1067007 BBa_K1067007]). Primers were design by the program [http://www.cbs.dtu.dk/services/PHUSER-2.0/web/ PHUSER] [5] so that we got a seamless assembly.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:PZA21-MCS TAT araBAD.png|680px|alt=Alt|"Hello World"" construct]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:PZA21-MCS TAT araBAD.png|680px|alt=Alt|"Hello World"" construct]]</div></td></tr>
</table>Helencookhttp://2013.igem.org/wiki/index.php?title=Team:DTU-Denmark/HelloWorld&diff=298759&oldid=prevHelencook: /* Introduction */2013-10-04T17:52:34Z<p><span class="autocomment">Introduction</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Introduction ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Introduction ==</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>‘Hello World!’ are the first words a programmer prints when learning a new programming language. <del class="diffchange diffchange-inline">In analogy to this </del>our team decided to do a ‘Hello World’ project in order to familiarize ourselves with lab techniques that we used later on to construct plasmids. Specifically we were performing PCR with uracil-containing primers, purifying PCR products and ligating them by means of USER cloning [1].</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>‘Hello World!’ are the first words a programmer prints when learning a new programming language. <ins class="diffchange diffchange-inline">Analogously, </ins>our team decided to do a ‘Hello World’ project in order to familiarize ourselves with lab techniques that we used later on to construct plasmids. Specifically we were performing PCR with uracil-containing primers, purifying PCR products and ligating them by means of USER cloning [1].</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since we are working with many periplasmic proteins, we wanted to try to target proteins to the periplasm. To do this, we used periplasmic signal peptides from the TAT and Sec pathways, and with a translational fusion of the signal peptide to GFP, we expressed GFP in the periplasm. Simultaneously, we expressed RFP in the cytoplasm as a background color, inspired by Skoog, Karl, et al. [2].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Since we are working with many periplasmic proteins, we wanted to try to target proteins to the periplasm. To do this, we used periplasmic signal peptides from the TAT and Sec pathways, and with a translational fusion of the signal peptide to GFP, we expressed GFP in the periplasm. Simultaneously, we expressed RFP in the cytoplasm as a background color, inspired by Skoog, Karl, et al. [2].</div></td></tr>
</table>Helencookhttp://2013.igem.org/wiki/index.php?title=Team:DTU-Denmark/HelloWorld&diff=298592&oldid=prevKrDa: /* Model of "Hello World" construct */2013-10-04T17:46:04Z<p><span class="autocomment">Model of "Hello World" construct</span></p>
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</table>KrDahttp://2013.igem.org/wiki/index.php?title=Team:DTU-Denmark/HelloWorld&diff=298558&oldid=prevKrDa at 17:45, 4 October 20132013-10-04T17:45:12Z<p></p>
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</table>KrDahttp://2013.igem.org/wiki/index.php?title=Team:DTU-Denmark/HelloWorld&diff=298543&oldid=prevKrDa: /* Model of "Hello World" construct */2013-10-04T17:44:22Z<p><span class="autocomment">Model of "Hello World" construct</span></p>
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