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Building off of a rapamycin induced split-luciferase system characterized by the 2015 Peking iGEM team, our construct consists of a promoter reporter system that looks at two downstream products, c-Myc and COX-2 (Peking iGEM Team 2015, 2015). These products are assembled into a construct composed of fusion proteins as well as split luciferase fragments (COX-2 - FRB - nLuc and c-Myc - FKBP - cLuc). In the presence of rapamycin, the interacting protein partners dimerize and subsequently cause luciferase to activate. In order to test this model, we created vectors of our constructs and inserted them into E. coli cells.
Our gene production construct is modeled to produce COX-2 and c-Myc, which we used to simulate conditions in human bodies when these proteins are overproduced, in the case of CRC. The construct consists of an Anderson promoter, a ribosomal binding site, a COX-2 gene/c-Myc gene, and a terminator.
Our promoter-reporter construct is built so that only when there is both COX-2 and c-Myc will the binding sites be able to come together and glow with the addition of rapamycin. The construct built to bind with COX-2 consists of a COX-2 promoter, FRB, n-Luc, and terminator. The construct built to bind with c-Myc consists of a c-Myc promoter, FKBP, c-Luc, and terminator.
Our promoter-reporter system consists of two separate constructs each with a gene-specific promoter attached to a non-specific binding site fused to a domain of split luciferase. One construct consists of a COX-2 promoter, a FRB domain, and n-Luc, and the other consists of a c-Myc promoter, a FKBP domain, and c-Luc. The split system is built so that only when the presence of both COX-2 and c-Myc is detected will the binding sites be able to come together with the addition of rapamycin and cause glowing.
In solution, our two gene constructs are able to simulate the conditions in the human body by producing proteins COX-2 and c-Myc. These proteins then interact with the promoter-reporter constructs by connecting to the non- specific binding sites, allowing the dimerization of the FKBP-Rapamycin-FRB complex. This then orients the split- luciferase so they can come together.
