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Team:Trieste/notebook8
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<div id="suicide" class="notebook_section"> | <div id="suicide" class="notebook_section"> | ||
<h2 class="notebook_title">Suicide System</h2> | <h2 class="notebook_title">Suicide System</h2> | ||
| - | We tried to ultimate the testing construct: we cloned the RBS_B0031-Tse2 toxin- TT_B0015 upstream the J23100 promoter-CymR-TT_B0015, with some issues. | + | We tried to ultimate the testing construct: we cloned the RBS_B0031-Tse2 toxin- TT_B0015 upstream the J23100 promoter-CymR-TT_B0015, with some issues.Team iGEM 2012 |
</div> | </div> | ||
<div id="antibody" class="notebook_section"> | <div id="antibody" class="notebook_section"> | ||
<h2 class="notebook_title">Antibody</h2> | <h2 class="notebook_title">Antibody</h2> | ||
| - | This week we used E. coli strain HB2151 to test pelB-SIP and pelB-scFv production. First, we transformed HB2151 with plasmids containing two versions of scFv. After 4 hour induction of recombinant bacterial cultures, we separated the periplasm content and then we proceeded with SDS-PAGE and Western blotting. On western blot, obtained this way, the bands corresponding to our proteins are well visible and aspecific signals are not present. | + | This week we used <i>E. coli</i> strain HB2151 to test pelB-SIP and pelB-scFv production. First, we transformed HB2151 with plasmids containing two versions of scFv. After 4 hour induction of recombinant bacterial cultures, we separated the periplasm content and then we proceeded with SDS-PAGE and Western blotting. On western blot, obtained this way, the bands corresponding to our proteins are well visible and aspecific signals are not present. |
</div> | </div> | ||
<div id="chassis" class="notebook_section"> | <div id="chassis" class="notebook_section"> | ||
| - | <h2 class="notebook_title"> | + | <h2 class="notebook_title">Cumate-Switch Regulation</h2> |
<u><b>CymR</b></u> | <u><b>CymR</b></u> | ||
| - | We ligated again the CymR upstream from the B0015 and then we cloned this whole fragment CymR-B0015 downstream from the J23100 in the | + | <br/> |
| - | </br> | + | We ligated again the CymR upstream from the B0015 and then we cloned this whole fragment CymR-B0015 downstream from the J23100 in the J61002 plasmid. We sequenced it and afterward we did the western blot and we saw that the protein CymR was produced. |
| + | <br/> | ||
| + | <br/> | ||
<u><b>T5 PROMOTER - CUMATE OPERATOR</b></u> | <u><b>T5 PROMOTER - CUMATE OPERATOR</b></u> | ||
| - | < | + | <br/> |
We decided to change strategy so we change the GFP E0240 and we chose a strongest one: I13504. We digested it with Xba/Pst and we ligated in the T5 operator-pSB1C3 Spe/Pst. After some problems with the ligation we find some positive colonies. | We decided to change strategy so we change the GFP E0240 and we chose a strongest one: I13504. We digested it with Xba/Pst and we ligated in the T5 operator-pSB1C3 Spe/Pst. After some problems with the ligation we find some positive colonies. | ||
</div> | </div> | ||
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<li><a href="https://2012.igem.org/Team:Trieste/notebook6">Week 6</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook6">Week 6</a></li> | ||
<li><a href="https://2012.igem.org/Team:Trieste/notebook7">Week 7</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook7">Week 7</a></li> | ||
| - | <li><a href="https://2012.igem.org/Team:Trieste/notebook8">Week 8</a></li> | + | <li class="select" ><a href="https://2012.igem.org/Team:Trieste/notebook8">Week 8</a></li> |
<li><a href="https://2012.igem.org/Team:Trieste/notebook9">Week 9</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook9">Week 9</a></li> | ||
<li><a href="https://2012.igem.org/Team:Trieste/notebook10">Week 10</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook10">Week 10</a></li> | ||
<li><a href="https://2012.igem.org/Team:Trieste/notebook11">Week 11</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook11">Week 11</a></li> | ||
<li><a href="https://2012.igem.org/Team:Trieste/notebook12">Week 12</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook12">Week 12</a></li> | ||
| + | <li><a href="https://2012.igem.org/Team:Trieste/notebooklast">Post European-Jamboree</a></li> | ||
</ul> | </ul> | ||
<img src="https://static.igem.org/mediawiki/2012/b/b0/Team_trieste.jpg" alt="Team iGEM 2012" id="igem_team" /> | <img src="https://static.igem.org/mediawiki/2012/b/b0/Team_trieste.jpg" alt="Team iGEM 2012" id="igem_team" /> | ||
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Follow us also: | Follow us also: | ||
<a href="https://www.facebook.com/IGEMUNITS?ref=ts" target="_blank"><img src="https://static.igem.org/mediawiki/2012/f/f4/Ico_fb.png" alt="Facebook" class="fb" /></a> | <a href="https://www.facebook.com/IGEMUNITS?ref=ts" target="_blank"><img src="https://static.igem.org/mediawiki/2012/f/f4/Ico_fb.png" alt="Facebook" class="fb" /></a> | ||
| - | <a href="https://twitter.com/ | + | <a href="https://twitter.com/iGEMTrieste" target="_blank"><img src="https://static.igem.org/mediawiki/2012/2/21/Ico_twitter.png" alt="twitter" class="tw" /></a> |
</div> | </div> | ||
</div> | </div> | ||
Latest revision as of 18:08, 26 October 2012
The Jolly JoCare
a safe probiotic platform for protein expression
Week 8
More
Suicide System
We tried to ultimate the testing construct: we cloned the RBS_B0031-Tse2 toxin- TT_B0015 upstream the J23100 promoter-CymR-TT_B0015, with some issues.Team iGEM 2012Antibody
This week we used E. coli strain HB2151 to test pelB-SIP and pelB-scFv production. First, we transformed HB2151 with plasmids containing two versions of scFv. After 4 hour induction of recombinant bacterial cultures, we separated the periplasm content and then we proceeded with SDS-PAGE and Western blotting. On western blot, obtained this way, the bands corresponding to our proteins are well visible and aspecific signals are not present.Cumate-Switch Regulation
CymRWe ligated again the CymR upstream from the B0015 and then we cloned this whole fragment CymR-B0015 downstream from the J23100 in the J61002 plasmid. We sequenced it and afterward we did the western blot and we saw that the protein CymR was produced.
T5 PROMOTER - CUMATE OPERATOR
We decided to change strategy so we change the GFP E0240 and we chose a strongest one: I13504. We digested it with Xba/Pst and we ligated in the T5 operator-pSB1C3 Spe/Pst. After some problems with the ligation we find some positive colonies.
