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Team:Trieste/notebook2
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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> | ||
| - | Finally we got by synthesis the antimicrobial peptide | + | Finally we got by synthesis the antimicrobial peptide LL 37 cathelicidin (BBa_K875009) in pEX-A plasmid and we amplified it through the transformation of DH5-alpha bacteria.</br> |
| - | </br> | + | |
The positive colonies were then digested EcoRI/SpeI, the fragment was eluted from the gel and then cut it again with EcoRI, because the pEX-A plasmid has another EcoRI restriction site downstream our fragment.</br> | The positive colonies were then digested EcoRI/SpeI, the fragment was eluted from the gel and then cut it again with EcoRI, because the pEX-A plasmid has another EcoRI restriction site downstream our fragment.</br> | ||
| - | + | ||
Then we did a column purification of the fragment and we successfully cloned the LL 37 into the pSB1C3 linearized backbone to make it ready for a potential submission.</br> | Then we did a column purification of the fragment and we successfully cloned the LL 37 into the pSB1C3 linearized backbone to make it ready for a potential submission.</br> | ||
| - | + | ||
| - | + | LL 37 -previously cut EcoRI/SpeI- was cloned upstream the TT_B0015 and the DH5alpha cells were transformed with the plasmid (LL 37-B0015). The positive colonies were inoculated and then cloned downstream the RBS_B0034.</br> | |
</br> | </br> | ||
We also cloned the T5LacOperator inside the pSB1C3 plasmid, making it ready for the submission. | We also cloned the T5LacOperator inside the pSB1C3 plasmid, making it ready for the submission. | ||
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<div id="antibody" class="notebook_section"> | <div id="antibody" class="notebook_section"> | ||
<h2 class="notebook_title">Antibody</h2> | <h2 class="notebook_title">Antibody</h2> | ||
| - | We cloned the synthetized DNA sequence rbs-pelB-SIP 54.6-6HIS upstream the double terminator B0015 and then we ligated successfully the whole fragment under T5LacO. We introduced the resulting plasmid into E. coli strain W3110 already transformed with plasmid p-REP 4 coding for T5Lac Repressor. | + | We cloned the synthetized DNA sequence rbs-pelB-SIP 54.6-6HIS upstream the double terminator B0015 and then we ligated successfully the whole fragment under T5LacO. We introduced the resulting plasmid into <i>E. coli</i> strain W3110 already transformed with plasmid p-REP 4 coding for T5Lac Repressor. |
</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> |
| - | + | We received the two plasmids that we had built and ordered. | |
| + | The first one contains the RBS - CymR - SV5 tag (730bp), the second one contains T5 promoter - Cumate operator (129 bp). | ||
</div> | </div> | ||
</div> | </div> | ||
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<ul id="sub_menu"> | <ul id="sub_menu"> | ||
<li><a href="https://2012.igem.org/Team:Trieste/notebook">Week 1</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook">Week 1</a></li> | ||
| - | <li><a href="https://2012.igem.org/Team:Trieste/notebook2">Week 2</a></li> | + | <li class="select" ><a href="https://2012.igem.org/Team:Trieste/notebook2">Week 2</a></li> |
<li><a href="https://2012.igem.org/Team:Trieste/notebook3">Week 3</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook3">Week 3</a></li> | ||
<li><a href="https://2012.igem.org/Team:Trieste/notebook4">Week 4</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/notebook4">Week 4</a></li> | ||
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<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 18:05, 26 October 2012
The Jolly JoCare
a safe probiotic platform for protein expression
Week 2
More
Suicide System
Finally we got by synthesis the antimicrobial peptide LL 37 cathelicidin (BBa_K875009) in pEX-A plasmid and we amplified it through the transformation of DH5-alpha bacteria. The positive colonies were then digested EcoRI/SpeI, the fragment was eluted from the gel and then cut it again with EcoRI, because the pEX-A plasmid has another EcoRI restriction site downstream our fragment. Then we did a column purification of the fragment and we successfully cloned the LL 37 into the pSB1C3 linearized backbone to make it ready for a potential submission. LL 37 -previously cut EcoRI/SpeI- was cloned upstream the TT_B0015 and the DH5alpha cells were transformed with the plasmid (LL 37-B0015). The positive colonies were inoculated and then cloned downstream the RBS_B0034. We also cloned the T5LacOperator inside the pSB1C3 plasmid, making it ready for the submission.Antibody
We cloned the synthetized DNA sequence rbs-pelB-SIP 54.6-6HIS upstream the double terminator B0015 and then we ligated successfully the whole fragment under T5LacO. We introduced the resulting plasmid into E. coli strain W3110 already transformed with plasmid p-REP 4 coding for T5Lac Repressor.Cumate-Switch Regulation
We received the two plasmids that we had built and ordered. The first one contains the RBS - CymR - SV5 tag (730bp), the second one contains T5 promoter - Cumate operator (129 bp).
