Suicide System

Finally we got by synthesis the antimicrobial peptide LL37 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).