Difference between revisions of "Part:BBa K2226000"

Revision as of 02:27, 28 October 2017

COX-2 promoter - FRB - Nluc398

COX-2 promoter, FRB rapamycin-binding domain (FRB) of human mTOR, fused to n-terminal of split luciferase (Nluc398), acts as one half of a promoter-reporter system, forms FRB-rapamycin-FKBP complex in prescence of rapamycin.

The FRB-Nluc part was taken from the 2015 Peking iGEM team, and we further characterized it by attaching a gene-specific promoter for COX-2. This altered the shape of the non-specific binding site to detect COX-2 proteins, acting as a biosensor.

In order to test the construct, we combined the COX-2 promoter-reporter construct with the c-Myc promoter-reporter construct in a solution with COX-2 and c-Myc gene constructs to model the conditions in the bodies of patients with CRC, where an excess of COX-2 and c-Myc protein is shed. As shown in the figures below, the combination of the COX-2 promoter-FRB-nLuc construct, c-Myc promoter-FKBP-cLuc construct in the presence of rapamycin and free-floating COX-2 and c-Myc protein successfully induced glowing.

In addition, to make sure the construct worked only with all components present (protein and rapamycin) we tested out multiple combinations with and without rapamycin, as well as each individual construct with rapamycin and without rapamycin on their own. The figures show that glowing was induced only with all the components present, proving that our construct can detect COX-2 and c-Myc proteins.

Above image showqualitative results demonstrate the constructs combining successfully in the presence of rapamycin when transformed in E. coli on plates and expanded in solution.

Sequence and Features