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Team:Trieste/notebook9
From 2012.igem.org
The Jolly JoCare
a safe probiotic platform for protein expression
Week 9
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Suicide System
We loaded the gel with the previously amplified -week 6- T5CumateOperator-RBS_B0034, eluted the fragment and then we tried to cut it, but it did not work as expected, probably because of the restriction enzymes do not work properly at the extremities. We approached to another strategy: we loaded the gel with the amplified, eluted the fragment and then amplified inside the pGEM vector. In this way, we were able to amplified the fragment with the primer pair SP6 and T7. The amplified were then cut with EcoRI/BamHI, heat inactivated and dephosphorylated at the extremities. It worked!! We were finally able to clone it upstream the T4 holin (BBa_K112000).Antibody
In order to assembly the final plasmid, we cloned LPP-OmpA-scFv downstream the constitutive promoter J23100. First, we amplified this plasmid in DH5alpha because Nissle are not competent enough to be transformed with a ligation. We tested its expression by SDS-PAGE and Western blotting and the results obtained testify that this protein is richly express into our bacteria. Eventually, we introduced our plasmid into E.coli Nissle.Cumate-Switch Regulation
CymRWe cloned the fragment J23100-CymR-B0015 downstream the same fragment to obtain the double CymR. The results were all negative so we made the same ligation over and over untill we obtained some positve colonies that seems to have the couple J23100-CymR-B0015 by PCR.
T5 PROMOTER - CUMATE OPERATOR
We cut them Xba/Pst and then we ligated this fragment in the J61002 plasmid Spe/Pst containing the J23100-CymR-B0015 fragment. We obtained some positive colonies.
