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Team:Trieste/notebook2
From 2012.igem.org
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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> |
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> | ||
Revision as of 21:16, 26 September 2012
The Jolly JoCare
a safe probiotic platform for protein expression
Week 2
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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).
