http://2013.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Results_summary&feed=atom&action=historyTeam:EPF Lausanne/Results summary - Revision history2024-03-29T15:02:29ZRevision history for this page on the wikiMediaWiki 1.16.5http://2013.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Results_summary&diff=338988&oldid=prevLeabernier at 18:12, 27 October 20132013-10-27T18:12:53Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Expression of streptavidin at the cell surface:'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Expression of streptavidin at the cell surface:'''</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Gibson assemblies of the three different streptavidin constructs worked and sequencing results matched with what expected. The growth curve of transformed E.Coli showed delayed growth, but bacteria still divide with an acceptable rate. The assay with a fluorescent biotin supposed to bind streptavidin gave some positive results (some bacteria appeared <del class="diffchange diffchange-inline">flurescent </del>when excited at the corresponding wavelength) but since the negative control also showed fluorescence, nothing could be proved. However, a Western blot against streptavidin showed bands at the expected size around 50 kDa of streptavidin, proving that it was expressed.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Gibson assemblies of the three different streptavidin constructs worked and sequencing results matched with what expected. The growth curve of transformed E.Coli showed delayed growth, but bacteria still divide with an acceptable rate. The assay with a fluorescent biotin supposed to bind streptavidin gave some positive results (some bacteria appeared <ins class="diffchange diffchange-inline">fluorescent </ins>when excited at the corresponding wavelength) but since the negative control also showed fluorescence, nothing could be proved. However, a Western blot against streptavidin showed bands at the expected size around 50 kDa of streptavidin, proving that it was expressed.</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>[[Image:Team-EPF-Lausanne_ISA5_biotin_3.10(RGB).jpg|thumb|300px|left| Figure 7: INP-streptavidin expressing cells from INP-strepta alive construct. Detection was made using fluorescently labeled biotin (green). Note that some cells were also positive on the negative control (competent cells), though they were less numerous.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_ISA5_biotin_3.10(RGB).jpg|thumb|300px|left| Figure 7: INP-streptavidin expressing cells from INP-strepta alive construct. Detection was made using fluorescently labeled biotin (green). Note that some cells were also positive on the negative control (competent cells), though they were less numerous.]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We inserted two pH-dependent and one constitutive promoter in front of a superfolded GFP sequence ( [http://parts.igem.org/Part:BBa_I746908 Biobrick BBa_I746916, Main page] ). All three promoters as well as the respective backbones were successfully isolated and amplified by PCR. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We inserted two pH-dependent and one constitutive promoter in front of a superfolded GFP sequence ( [http://parts.igem.org/Part:BBa_I746908 Biobrick BBa_I746916, Main page] ). All three promoters as well as the respective backbones were successfully isolated and amplified by PCR. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The Gibson assemblies also worked and the sequencing results showed a 100% match between the inserted promoters and the reference sequence.<BR></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The Gibson assemblies also worked and the sequencing results showed a 100% match between the inserted promoters and the reference sequence.<BR></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Fluorescence measurements with the <del class="diffchange diffchange-inline">PlateReader </del>were not conclusive since a lot of cells died in acidic pH, supposed to activate the promoter. However, even if we were not able to prove that acidic pH triggers expression of GFP, fluorescence could be seen under the microscope. This indicated that the promoters are functional. The constitutive promoter worked as expected, inducing the expression of superfolded GFP strongly. We used the fact that those cells' fluorescence could be seen by the naked eye and put the plasmid in our human practice kit ([https://2013.igem.org/Team:EPF_Lausanne/Kit Our Kit ]).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Fluorescence measurements with the <ins class="diffchange diffchange-inline">Plate Reader </ins>were not conclusive since a lot of cells died in acidic pH, supposed to activate the promoter. However, even if we were not able to prove that acidic pH triggers expression of GFP, fluorescence could be seen under the microscope. This indicated that the promoters are functional. The constitutive promoter worked as expected, inducing the expression of superfolded GFP strongly. We used the fact that those cells' fluorescence could be seen by the naked eye and put the plasmid in our human practice kit ([https://2013.igem.org/Team:EPF_Lausanne/Kit Our Kit ]).</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image: Team-EPFL-Lausanne PCR_1.1+1.2+1.3_BB.jpg|thumb|200px|left|Figure 9: 0.8% Gel, PCRs of the three backbones ]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image: Team-EPFL-Lausanne PCR_1.1+1.2+1.3_BB.jpg|thumb|200px|left|Figure 9: 0.8% Gel, PCRs of the three backbones ]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image: Team-EPFL-Lausanne PlateReader.jpg|thumb|500px|right|Figure 10: Plate used for the <del class="diffchange diffchange-inline">PlateReader </del>experiment. The cell marked in a black rectangle are the ones containing the constitutive promoter.]]<br><br><br><br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image: Team-EPFL-Lausanne PlateReader.jpg|thumb|500px|right|Figure 10: Plate used for the <ins class="diffchange diffchange-inline">Plate Reader </ins>experiment. The cell marked in a black rectangle are the ones containing the constitutive promoter.]]<br><br><br><br><br></div></td></tr>
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</table>Leabernierhttp://2013.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Results_summary&diff=316949&oldid=prevLeabernier at 03:47, 5 October 20132013-10-05T03:47:54Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Expression of streptavidin at the cell surface:'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Expression of streptavidin at the cell surface:'''</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Gibson assemblies of the three different streptavidin constructs worked and sequencing results matched with what expected. The growth curve of transformed E.Coli showed delayed growth, but bacteria still divide with an acceptable rate. The assay with a fluorescent biotin supposed to bind streptavidin gave some positive results (some bacteria appeared flurescent when excited at the corresponding <del class="diffchange diffchange-inline">wavelenght</del>) but since the negative control also showed fluorescence, nothing could be proved. However, a Western blot against streptavidin showed bands at the expected size around 50 kDa of streptavidin, proving that it was expressed.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Gibson assemblies of the three different streptavidin constructs worked and sequencing results matched with what expected. The growth curve of transformed E.Coli showed delayed growth, but bacteria still divide with an acceptable rate. The assay with a fluorescent biotin supposed to bind streptavidin gave some positive results (some bacteria appeared flurescent when excited at the corresponding <ins class="diffchange diffchange-inline">wavelength</ins>) but since the negative control also showed fluorescence, nothing could be proved. However, a Western blot against streptavidin showed bands at the expected size around 50 kDa of streptavidin, proving that it was expressed.</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>[[Image:Team-EPF-Lausanne_ISA5_biotin_3.10(RGB).jpg|thumb|300px|left| Figure 7: INP-streptavidin expressing cells from INP-strepta alive construct. Detection was made using fluorescently labeled biotin (green). Note that some cells were also positive on the negative control (competent cells), though they were less numerous.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_ISA5_biotin_3.10(RGB).jpg|thumb|300px|left| Figure 7: INP-streptavidin expressing cells from INP-strepta alive construct. Detection was made using fluorescently labeled biotin (green). Note that some cells were also positive on the negative control (competent cells), though they were less numerous.]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Sensing:==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Sensing:==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We inserted two pH-dependent and one constitutive promoter in front of a superfolded GFP sequence ( [http://parts.igem.org/Part:BBa_I746908 Biobrick BBa_I746916, Main page] ). All three promoters as well as the respective backbones were successfully isolated and amplified by PCR. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We inserted two pH-dependent and one constitutive promoter in front of a superfolded GFP sequence ( [http://parts.igem.org/Part:BBa_I746908 Biobrick BBa_I746916, Main page] ). All three promoters as well as the respective backbones were successfully isolated and amplified by PCR. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The Gibson assemblies also worked and the sequencing results showed a 100% match between the inserted promoters and the <del class="diffchange diffchange-inline">refererence </del>sequence.<BR></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The Gibson assemblies also worked and the sequencing results showed a 100% match between the inserted promoters and the <ins class="diffchange diffchange-inline">reference </ins>sequence.<BR></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Fluorescence measurements with the PlateReader were not conclusive since a lot of cells died in acidic pH, supposed to activate the promoter. However, even if we were not able to prove that acidic pH triggers expression of GFP, fluorescence could be seen under the microscope. This indicated that the promoters are functional. The constitutive promoter worked as expected, inducing the expression of superfolded GFP strongly. We used the fact that those cells' fluorescence could be seen by the naked eye and put the plasmid in our human practice kit ([https://2013.igem.org/Team:EPF_Lausanne/Kit Our Kit ]).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Fluorescence measurements with the PlateReader were not conclusive since a lot of cells died in acidic pH, supposed to activate the promoter. However, even if we were not able to prove that acidic pH triggers expression of GFP, fluorescence could be seen under the microscope. This indicated that the promoters are functional. The constitutive promoter worked as expected, inducing the expression of superfolded GFP strongly. We used the fact that those cells' fluorescence could be seen by the naked eye and put the plasmid in our human practice kit ([https://2013.igem.org/Team:EPF_Lausanne/Kit Our Kit ]).</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image: Team-EPFL-Lausanne PCR_1.1+1.2+1.3_BB.jpg|thumb|200px|left|Figure 9: 0.8% Gel, PCRs of the three backbones ]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image: Team-EPFL-Lausanne PCR_1.1+1.2+1.3_BB.jpg|thumb|200px|left|Figure 9: 0.8% Gel, PCRs of the three backbones ]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Effector:==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Effector:==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>With the exception of MMP9, the Gibson Assemblies worked for both Gelatinase GelE and MMP2. The primers that were designed had introduced a stop codon upstream of GFP. But since we had planned to do constructs with and without GFP attached to the gelatinase, we continued our experiments. The Western Blot with an anti-His tag antibody did not work. The His-Tag may be hidden in the protein, contain a <del class="diffchange diffchange-inline">mutaion </del>or could simply not bind to the nickel columns as they were stored improperly. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>With the exception of MMP9, the Gibson Assemblies worked for both Gelatinase GelE and MMP2. The primers that were designed had introduced a stop codon upstream of GFP. But since we had planned to do constructs with and without GFP attached to the gelatinase, we continued our experiments. The Western Blot with an anti-His tag antibody did not work. The His-Tag may be hidden in the protein, contain a <ins class="diffchange diffchange-inline">mutation </ins>or could simply not bind to the nickel columns as they were stored improperly. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Overall:==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Overall:==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Globally, we can say that our cloning was successful. Most of the Gibson assemblies worked and we had no particular troubles growing the resulting transformed bacteria. The most delicate part was the characterization of our parts with <del class="diffchange diffchange-inline">functionnal </del>assays. Though some experiments didn't allow us to make conclusions, a lot of parts showed encouraging results. They would maybe need to be studied in more detail for further improvement. The nanoparticle module worked very well. Our positive results would allow to try to load real drugs in a next nanoparticles batch.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Globally, we can say that our cloning was successful. Most of the Gibson assemblies worked and we had no particular troubles growing the resulting transformed bacteria. The most delicate part was the characterization of our parts with <ins class="diffchange diffchange-inline">functional </ins>assays. Though some experiments didn't allow us to make conclusions, a lot of parts showed encouraging results. They would maybe need to be studied in more detail for further improvement. The nanoparticle module worked very well. Our positive results would allow to try to load real drugs in a next nanoparticles batch.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This project was ambitious and was almost achieved, and we are really proud to share our experience with the iGEM community!</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This project was ambitious and was almost achieved, and we are really proud to share our experience with the iGEM community!</div></td></tr>
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</table>Leabernierhttp://2013.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Results_summary&diff=315680&oldid=prevSandelisa90: /* Effector: */2013-10-05T03:07:44Z<p><span class="autocomment">Effector:</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Effector:==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Effector:==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>With the exception of MMP9, the Gibson Assemblies worked for both Gelatinase GelE and MMP2. The primers that were designed had introduced a stop codon upstream of GFP. But since we had planned to do constructs with and without GFP attached to the gelatinase, we continued our experiments. The Western Blot with an anti-His tag antibody did not work. The His-Tag <del class="diffchange diffchange-inline">it </del>may be hidden in the protein, contain a mutaion or could simply not bind to the nickel columns as they were stored improperly. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>With the exception of MMP9, the Gibson Assemblies worked for both Gelatinase GelE and MMP2. The primers that were designed had introduced a stop codon upstream of GFP. But since we had planned to do constructs with and without GFP attached to the gelatinase, we continued our experiments. The Western Blot with an anti-His tag antibody did not work. The His-Tag may be hidden in the protein, contain a mutaion or could simply not bind to the nickel columns as they were stored improperly. </div></td></tr>
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</table>Sandelisa90http://2013.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Results_summary&diff=315601&oldid=prevSandelisa90: /* Nanoparticles: */2013-10-05T03:04:48Z<p><span class="autocomment">Nanoparticles:</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_results_NPs_DLS1.jpg|thumb|250px|left|Figure 1: DLS experiment results of the first nanoparticles we obtained: their mean diameter is a bit below 200 nm.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_results_NPs_DLS1.jpg|thumb|250px|left|Figure 1: DLS experiment results of the first nanoparticles we obtained: their mean diameter is a bit below 200 nm.]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_results_ELISA-like2.jpg|thumb|<del class="diffchange diffchange-inline">210px</del>|left|Figure 2: ELISA-like assay. The high absorbance in wells B and H (containing biotinylated nanoparticles) was quantitatively detected using a plate reader. It showed that the nanoparticles were well biotinylated.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_results_ELISA-like2.jpg|thumb|<ins class="diffchange diffchange-inline">250px</ins>|left|Figure 2: ELISA-like assay. The high absorbance in wells B and H (containing biotinylated nanoparticles) was quantitatively detected using a plate reader. It showed that the nanoparticles were well biotinylated.]]</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>[[Image:Team-EPF-Lausanne_results_confocal_positive.jpg|thumb|200px|left| Figure 3: Confocal microscopy image showing outer CY5 labeling of the nanoparticles. The size of those nanoparticles is around 200 nm, which corresponds to the previous DLS characterization.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_results_confocal_positive.jpg|thumb|200px|left| Figure 3: Confocal microscopy image showing outer CY5 labeling of the nanoparticles. The size of those nanoparticles is around 200 nm, which corresponds to the previous DLS characterization.]]</div></td></tr>
</table>Sandelisa90http://2013.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Results_summary&diff=315586&oldid=prevSandelisa90: /* Nanoparticles: */2013-10-05T03:04:28Z<p><span class="autocomment">Nanoparticles:</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>These experiments show that two cargo transport strategies are possible: an external labeling (the one we used with CY5) or an internal loading (with FITC-Dextran and rGFP).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>These experiments show that two cargo transport strategies are possible: an external labeling (the one we used with CY5) or an internal loading (with FITC-Dextran and rGFP).</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_results_NPs_DLS1.jpg|thumb|<del class="diffchange diffchange-inline">240px</del>|left|Figure 1: DLS experiment results of the first nanoparticles we obtained: their mean diameter is a bit below 200 nm.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_results_NPs_DLS1.jpg|thumb|<ins class="diffchange diffchange-inline">250px</ins>|left|Figure 1: DLS experiment results of the first nanoparticles we obtained: their mean diameter is a bit below 200 nm.]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_results_ELISA-like2.jpg|thumb|<del class="diffchange diffchange-inline">240px</del>|left|Figure 2: ELISA-like assay. The high absorbance in wells B and H (containing biotinylated nanoparticles) was quantitatively detected using a plate reader. It showed that the nanoparticles were well biotinylated.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_results_ELISA-like2.jpg|thumb|<ins class="diffchange diffchange-inline">210px</ins>|left|Figure 2: ELISA-like assay. The high absorbance in wells B and H (containing biotinylated nanoparticles) was quantitatively detected using a plate reader. It showed that the nanoparticles were well biotinylated.]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_results_confocal_positive.jpg|thumb|<del class="diffchange diffchange-inline">240px</del>|left| Figure 3: Confocal microscopy image showing outer CY5 labeling of the nanoparticles. The size of those nanoparticles is around 200 nm, which corresponds to the previous DLS characterization.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_results_confocal_positive.jpg|thumb|<ins class="diffchange diffchange-inline">200px</ins>|left| Figure 3: Confocal microscopy image showing outer CY5 labeling of the nanoparticles. The size of those nanoparticles is around 200 nm, which corresponds to the previous DLS characterization.]]</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>[[Image:Team-EPF-Lausanne_results_NPs_FITC.jpg|thumb|240px|left| Figure 4: Fluorescent microscopy of the FITC-Dextran loaded nanoparticles. In contrast to the rGFP-loaded ones, their cargo stay inside. Those nanoparticles have been successfully characterized and biotinylated.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_results_NPs_FITC.jpg|thumb|240px|left| Figure 4: Fluorescent microscopy of the FITC-Dextran loaded nanoparticles. In contrast to the rGFP-loaded ones, their cargo stay inside. Those nanoparticles have been successfully characterized and biotinylated.]]</div></td></tr>
</table>Sandelisa90http://2013.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Results_summary&diff=315561&oldid=prevSandelisa90: /* Cell surface expression: */2013-10-05T03:03:43Z<p><span class="autocomment">Cell surface expression:</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_ISA5_biotin_3.10(RGB).jpg|thumb|300px|left| Figure 7: INP-streptavidin expressing cells from INP-strepta alive construct. Detection was made using fluorescently labeled biotin (green). Note that some cells were also positive on the negative control (competent cells), though they were less numerous.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_ISA5_biotin_3.10(RGB).jpg|thumb|300px|left| Figure 7: INP-streptavidin expressing cells from INP-strepta alive construct. Detection was made using fluorescently labeled biotin (green). Note that some cells were also positive on the negative control (competent cells), though they were less numerous.]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_WB1.jpg|thumb|<del class="diffchange diffchange-inline">400px</del>|left| Figure 8: Western blot made from total protein of INP-strepta (from all three constructs) transformed cells. Though there is much unspecific noise, there are bands at the right size.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_WB1.jpg|thumb|<ins class="diffchange diffchange-inline">375px</ins>|left| Figure 8: Western blot made from total protein of INP-strepta (from all three constructs) transformed cells. Though there is much unspecific noise, there are bands at the right size.]]</div></td></tr>
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</table>Sandelisa90http://2013.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Results_summary&diff=315551&oldid=prevSandelisa90: /* Cell surface expression: */2013-10-05T03:03:19Z<p><span class="autocomment">Cell surface expression:</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This fluorescence microscopy image shows that YFP direct excitation signal colocalizes with YFP-antibody signal, meaning that the protein is not only at the membrane, but at the outer one.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This fluorescence microscopy image shows that YFP direct excitation signal colocalizes with YFP-antibody signal, meaning that the protein is not only at the membrane, but at the outer one.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_INP-WFP-merged-3pics.jpg|thumb|<del class="diffchange diffchange-inline">600px</del>|left| Figure 6: INP-YFP expressing cells A)detection by WFP excitation (514nm); B) detection by biotinylated anti-YFP antibody and avidin daylight (650nm); C) Merged images show colocalization, proving that the fusion protein is expressed at the outer membrane.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_INP-WFP-merged-3pics.jpg|thumb|<ins class="diffchange diffchange-inline">700px</ins>|left| Figure 6: INP-YFP expressing cells A)detection by WFP excitation (514nm); B) detection by biotinylated anti-YFP antibody and avidin daylight (650nm); C) Merged images show colocalization, proving that the fusion protein is expressed at the outer membrane.]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br><br><br><br><br><br><br><br><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br><br><br><br><br><br><br><br><br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_ISA5_biotin_3.10(RGB).jpg|thumb|300px|left| Figure 7: INP-streptavidin expressing cells from INP-strepta alive construct. Detection was made using fluorescently labeled biotin (green). Note that some cells were also positive on the negative control (competent cells), though they were less numerous.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_ISA5_biotin_3.10(RGB).jpg|thumb|300px|left| Figure 7: INP-streptavidin expressing cells from INP-strepta alive construct. Detection was made using fluorescently labeled biotin (green). Note that some cells were also positive on the negative control (competent cells), though they were less numerous.]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_WB1.jpg|thumb|<del class="diffchange diffchange-inline">300px</del>|left| Figure 8: Western blot made from total protein of INP-strepta (from all three constructs) transformed cells. Though there is much unspecific noise, there are bands at the right size.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_WB1.jpg|thumb|<ins class="diffchange diffchange-inline">400px</ins>|left| Figure 8: Western blot made from total protein of INP-strepta (from all three constructs) transformed cells. Though there is much unspecific noise, there are bands at the right size.]]</div></td></tr>
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</table>Sandelisa90http://2013.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Results_summary&diff=315528&oldid=prevSandelisa90: /* Cell surface expression: */2013-10-05T03:02:29Z<p><span class="autocomment">Cell surface expression:</span></p>
<table style="background-color: white; color:black;">
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<td colspan='2' style="background-color: white; color:black;">Revision as of 03:02, 5 October 2013</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Characterization of an existing part:'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Characterization of an existing part:'''</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The part BBa_K523013 (INP-YFP construct to export YFP at the membrane) had been characterized only by a comparison of pellet and supernatant fluorescence after centrifugation. <br>We wanted to characterize it better to be sure that it was expressed on the outer membrane. We successfully showed that the <del class="diffchange diffchange-inline">YFP-INP </del>was expressed at the membrane.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The part BBa_K523013 (INP-YFP construct to export YFP at the membrane) had been characterized only by a comparison of pellet and supernatant fluorescence after centrifugation. <br>We wanted to characterize it better to be sure that it was expressed on the outer membrane. We successfully showed that the <ins class="diffchange diffchange-inline">INP_YFP fusion protein </ins>was expressed at the membrane.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>This fluorescence microscopy image shows that YFP direct excitation signal colocalizes with YFP-<del class="diffchange diffchange-inline">antibodysignal</del>, meaning that the protein is not only at the membrane, but at the outer one.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>This fluorescence microscopy image shows that YFP direct excitation signal colocalizes with YFP-<ins class="diffchange diffchange-inline">antibody signal</ins>, meaning that the protein is not only at the membrane, but at the outer one.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_INP-WFP-merged-3pics.jpg|thumb|600px|left| Figure 6: INP-YFP expressing cells A)detection by WFP excitation (514nm); B) detection by biotinylated anti-YFP antibody and avidin daylight (650nm); C) Merged images show colocalization, proving that the fusion protein is expressed at the outer membrane.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_INP-WFP-merged-3pics.jpg|thumb|600px|left| Figure 6: INP-YFP expressing cells A)detection by WFP excitation (514nm); B) detection by biotinylated anti-YFP antibody and avidin daylight (650nm); C) Merged images show colocalization, proving that the fusion protein is expressed at the outer membrane.]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br><br><br><br><br><br><br><br><br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><br><br><br><br><br><br><br><br><br><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Expression of streptavidin at the cell surface:'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Expression of streptavidin at the cell surface:'''</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Gibson assemblies of the three different streptavidin constructs worked<del class="diffchange diffchange-inline">. </del>and sequencing results matched with what expected. The growth curve of transformed E.Coli showed delayed growth, but bacteria still divide with an acceptable rate. The assay with a fluorescent biotin supposed to bind streptavidin gave some positive results (some bacteria appeared flurescent when excited at the corresponding wavelenght) but since the negative control also showed fluorescence, nothing could be proved. However, a Western blot against streptavidin showed bands at the expected size <del class="diffchange diffchange-inline">(53 </del>kDa<del class="diffchange diffchange-inline">) </del>of streptavidin, proving that it was expressed.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Gibson assemblies of the three different streptavidin constructs worked and sequencing results matched with what expected. The growth curve of transformed E.Coli showed delayed growth, but bacteria still divide with an acceptable rate. The assay with a fluorescent biotin supposed to bind streptavidin gave some positive results (some bacteria appeared flurescent when excited at the corresponding wavelenght) but since the negative control also showed fluorescence, nothing could be proved. However, a Western blot against streptavidin showed bands at the expected size <ins class="diffchange diffchange-inline">around 50 </ins>kDa of streptavidin, proving that it was expressed.</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>[[Image:Team-EPF-Lausanne_ISA5_biotin_3.10(RGB).jpg|thumb|300px|left| Figure 7: INP-streptavidin expressing cells from INP-strepta alive construct. Detection was made using fluorescently labeled biotin (green). Note that some cells were also positive on the negative control (competent cells), though they were less numerous.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Team-EPF-Lausanne_ISA5_biotin_3.10(RGB).jpg|thumb|300px|left| Figure 7: INP-streptavidin expressing cells from INP-strepta alive construct. Detection was made using fluorescently labeled biotin (green). Note that some cells were also positive on the negative control (competent cells), though they were less numerous.]]</div></td></tr>
</table>Sandelisa90http://2013.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Results_summary&diff=315395&oldid=prevSandelisa90: /* Cell surface expression: */2013-10-05T02:58:24Z<p><span class="autocomment">Cell surface expression:</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 02:58, 5 October 2013</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Characterization of an existing part:'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Characterization of an existing part:'''</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The part BBa_K523013 (INP-YFP construct to export YFP at the membrane) had been characterized only by a comparison of pellet and supernatant fluorescence after centrifugation. We wanted to characterize it better to be sure that it was expressed on the outer membrane. We successfully showed that the YFP-INP was expressed at the membrane.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The part BBa_K523013 (INP-YFP construct to export YFP at the membrane) had been characterized only by a comparison of pellet and supernatant fluorescence after centrifugation. <ins class="diffchange diffchange-inline"><br></ins>We wanted to characterize it better to be sure that it was expressed on the outer membrane. We successfully showed that the YFP-INP was expressed at the membrane.</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>This fluorescence microscopy image shows that YFP direct excitation signal colocalizes with YFP-antibodysignal, meaning that the protein is not only at the membrane, but at the outer one.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This fluorescence microscopy image shows that YFP direct excitation signal colocalizes with YFP-antibodysignal, meaning that the protein is not only at the membrane, but at the outer one.</div></td></tr>
</table>Sandelisa90http://2013.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Results_summary&diff=313526&oldid=prevSophierivara: /* Overall: */2013-10-05T01:51:47Z<p><span class="autocomment">Overall:</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Overall:==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Overall:==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">We </del>can say that <del class="diffchange diffchange-inline">except minor problems, </del>cloning <del class="diffchange diffchange-inline">succedeed well</del>. <del class="diffchange diffchange-inline">The </del>Gibson assemblies <del class="diffchange diffchange-inline">globally </del>worked <del class="diffchange diffchange-inline">out </del>and we had no <del class="diffchange diffchange-inline">trouble </del>growing the resulting transformed bacteria. The most delicate part was the characterization of our parts with functionnal assays. <del class="diffchange diffchange-inline">However</del>, a lot of parts showed encouraging results <del class="diffchange diffchange-inline">but </del>would maybe need to be studied in more detail. The <del class="diffchange diffchange-inline">naoparticles is the part that </del>worked <del class="diffchange diffchange-inline">out </del>well<del class="diffchange diffchange-inline">, </del>nanoparticles <del class="diffchange diffchange-inline">could be synthetized and loaded</del>. This project was ambitious and was almost achieved, and we are really proud <del class="diffchange diffchange-inline">of sharing </del>our <del class="diffchange diffchange-inline">results </del>with the iGEM community!</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Globally, we </ins>can say that <ins class="diffchange diffchange-inline">our </ins>cloning <ins class="diffchange diffchange-inline">was successful</ins>. <ins class="diffchange diffchange-inline">Most of the </ins>Gibson assemblies worked and we had no <ins class="diffchange diffchange-inline">particular troubles </ins>growing the resulting transformed bacteria. The most delicate part was the characterization of our parts with functionnal assays. <ins class="diffchange diffchange-inline">Though some experiments didn't allow us to make conclusions</ins>, a lot of parts showed encouraging results<ins class="diffchange diffchange-inline">. They </ins>would maybe need to be studied in more detail <ins class="diffchange diffchange-inline">for further improvement</ins>. The <ins class="diffchange diffchange-inline">nanoparticle module </ins>worked <ins class="diffchange diffchange-inline">very </ins>well<ins class="diffchange diffchange-inline">. Our positive results would allow to try to load real drugs in a next </ins>nanoparticles <ins class="diffchange diffchange-inline">batch</ins>.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>This project was ambitious and was almost achieved, and we are really proud <ins class="diffchange diffchange-inline">to share </ins>our <ins class="diffchange diffchange-inline">experience </ins>with the iGEM community!</div></td></tr>
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