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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.Chassis
CymR We 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.
