![](https://parts.igem.org/images/partbypart/icon_coding.png)
Coding
Part:BBa_K1965046
Designed by: Katja Leben Group: iGEM16_Slovenia (2016-10-17)
Revision as of 17:36, 18 October 2016 by NinaJerala (Talk | contribs)
Myc:B:nLuc
This construct consists of the N-terminal part of the split luciferase attached to the coiled coil B. The sequence for the coiled coil B was taken from the Shekhawat et al. (1). The construct also features a Myc protein tag on the N-terminus. Coiled coil B dimerizes with the coiled coil A, which was also described in Shekhawat et al.. If the A coil is fused to the C-terminal part of the split luciferase (BBa_K1965045), the luciferase can reassemble and regain its activity through the dimerization of the coiled coil pair A and B (2A) [1] .
![](https://static.igem.org/mediawiki/2016/5/5f/T--Slovenia--4.12.8.png)
We designed different destabilized coils from our coiled coil pair AP4 and P3; Ile and Leu were substituted with Ala at the a and/or d position of the first and/or second heptad of P3mS (a more soluble variant of the original P3).
![](https://static.igem.org/mediawiki/2016/1/16/T--Slovenia--4.12.1.png)
HEK293T cells were transfected with plasmids, coding for the appropriate coiled coil constructs. 24 h after transfection cells were lysed and luciferase activity was determined with the dual luciferase assay. (A) A and B coiled-coils, fused the split firefly luciferase spontaneously interact and reconstitute firefly luciferase. (B) A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A auto-inhibitory coiled coils reconstitute activity of firefly luciferase upon cleavage by TEV and PPV proteases. Successive luciferase reconstitution is observed only at high amounts of plasmids coding for the A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A constructs.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 229
Illegal NgoMIV site found at 1573
Illegal NgoMIV site found at 1594
Illegal AgeI site found at 1297 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1479
[edit]
Categories
Parameters
None |