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Team:Trieste/notebook4
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The Jolly JoCare
a safe probiotic platform for protein expression
Week 4
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Suicide System
Due to the negative results of the previous week , we decided to use another toxin from the registry, that we have already extracted from iGEM kit and amplified it through the transformation of DH5-alpha competent cells: Tse2 toxin (BB_K314200). We began to create our toxin construct, cutting Tse2 Toxin (EcoRI/SpeI) to clone it inside the double terminator BB_B0015, previously cut with EcoRI/XbaI. Unfortunately we did not obtain a positive colony, so we tried different cloning approaches.Antibody
We replaced pelB leader sequence with LPP-OmpA into the SIP fragment in order to attach the SIP on the bacterial surface. Then we cloned it downstream TLacO promoter. This construct was also tested in W3110 with p-REP 4. We induced its expression with IPTG (1mM). Following the same protocol, and we analysed the induced culture by Western blotting. Our fusion protein is being expressed.Chassis
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