Part:BBa_K2226005
c-Myc promoter - FKBP - Cluc394
c-Myc promoter, FK-506-binding protein 12 fused to c-terminal of split luciferase (Cluc394), acts as one half of a promoter-reporter system, forms FRB-rapamycin-FKBP complex in presence of rapamycin.
The FKBP-Cluc part was taken from the 2015 Peking iGEM team, and we further characterized it by attaching a gene-specific promoter for c-Myc gene. This altered the shape of the non-specific binding site to detect c-Myc 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 45
Illegal NheI site found at 68 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 688
Illegal AgeI site found at 827 - 1000COMPATIBLE WITH RFC[1000]
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