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Team:Trieste/notebook8
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The Jolly JoCare
a safe probiotic platform for protein expression
Week 8
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Suicide System
We tried to ultimate the testing construct: we cloned the RBS_B0031-Tse2 toxin- TT_B0015 upstream the J23100 promoter-CymR-TT_B0015, with some issues.Antibody
This week we used E. coli strain HB2151 to test pelB-SIP and pelB-scFv production. First, we transformed HB2151 with plasmids containing two versions of scFv. After 4 hour induction of recombinant bacterial cultures, we separated the periplasm content and then we proceeded with SDS-PAGE and Western blotting. On western blot, obtained this way, the bands corresponding to our proteins are well visible and aspecific signals are not present.Cumate-Switch Regulation
CymRWe ligated again the CymR upstream from the B0015 and then we cloned this whole fragment CymR-B0015 downstream from the J23100 in the J61002 plasmid. We sequenced it and afterward we did the western blot and we saw that the protein CymR was produced.
T5 PROMOTER - CUMATE OPERATOR
We decided to change strategy so we change the GFP E0240 and we chose a strongest one: I13504. We digested it with Xba/Pst and we ligated in the T5 operator-pSB1C3 Spe/Pst. After some problems with the ligation we find some positive colonies.
