Team:Fatih-Medical/Sherlocoli/Assembly

There are seven subparts in order to establish the designed system in this module, certainly. We pursued the goal of assembling those subparts via using 3A assembly method.

In Sherlocoli module, there are four composite parts whose functions varies in the mechanism: two antibody regions on the transmembrane protein which provides antigen binding domains and which possesses a transfection domain consisting of N and C terminuses of TEV protease (BBa_K772001 and BBa_K772002)and four different cleavable signal trigger protein complexes. (BBa_K772006/7/8/9)

The transmembrane protein docks of binding and cleavage domains are designed as a whole gene sequence which has no genomic scars because of the lack of using restriction digestion. These two proteins are fixed together via 3A Assembly method and transformed into the E.coli. There are pLac constitutive promoter and appropriate RBS on the upstream region of protein sequences in order to have maximum synthesis speed. The Co-IP assay will be carried out with this part. The result of this experiment will hopefully provide information about how our antigen binding cassette reacts to the transfection after being exposed to the tumor cells. ( BBa_K772100 )

In order to measure the TEV protease effect on the anchor-inducer proteins (signal trigger complexes) we designed an alternative reporter device which uses the FRET effect (the phenomenon which bases on light reflecting effect of cyan fluorescent protein towards the yellow fluorescent protein that is fixed to it.) pLac promoter and RBS will be added on the upstream region of the part to achieve constitutive production. It is hoped to have an idea about how the cleavage by TEV protease will be concluded after the transfection of the enzyme. (BBa_K772003)