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Team:Trieste/notebook6
From 2012.igem.org
The Jolly JoCare
a safe probiotic platform for protein expression
Week 6
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Suicide System
Holin is defined as a Bglbrick part, that means it doesn't have the standard restriction sites. We transformed the standard Biobrick we needed to use, in Bglbrick by PCR. The promoter T5CumateOperator(BBa_K875001) was cut EcoRI/SpeI, the fragment was eluted from the gel and then cloned upstream the RBS_B0034, into the pSB1A2 linearized backbone. We screened the colonies by PCR with the standard VF2 and VR primers and the positive ones were inoculated; we amplified the miniprep by PCR with particular primers with the EcoRI, BglII, BamHI and XhoI restriction sites.Antibody
The sequences arrived, and both scFv versions (pelB-scFv and LPP-scFv) are correct, so we continued with our work. Afer different attempts, we finally ligated both scFv versions under T5LacO. We amplified the resulting plasmid in DH5L and introduced it into W3110 (with p-REP 4) which we will use for future analysis.Cumate-Switch Regulation
CymRWe ligate the CymR-B0015 X/P in the plasmid J61002 (treated with SAP) downstream the J23100. We proceed in many directions because the cloning of CymR-B0015 downstream J23100 was very difficult: we repeated the ligation with the same plasmid but with different treatment :
-elution and SAP
-elution without SAP
-ligation without gel purification.
We obtained some positive clones that we digest E/P for analysis. So we digested and ligated the right clones together. We obtain the double copy J23100-CymR-B0015.
T5 PROMOTER - CUMATE OPERATOR
We started again from the T5 promoter-E0240. We cloned T5 promoter-E0240 downstream j23100. We obtained positive clones but they didn't work.
