Difference between revisions of "Part:BBa K2226000"

Latest revision as of 11:15, 31 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, modifying the shape.

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 when all where components present (gene constructs, promoter-reporter system, rapamycin) we tested out multiple combinations where certain components to show proof of concept. 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 45
Illegal NheI site found at 68
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 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.
+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, modifying the shape.
-By attaching the gene-specific promoter for COX-2 onto the
 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
+In addition, to make sure the construct worked only when all where components present (gene constructs, promoter-reporter system, rapamycin) we tested out multiple combinations where certain components to show proof of concept. 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.
-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.
 https://static.igem.org/mediawiki/parts/e/e9/Results_plates.png