Difference between revisions of "Part:BBa K1965049"
 
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<p>This construct consists of a C-terminal part of the split luciferase that is fused to the P5 coiled coil (<ref>1</ref>) via a linker which contains the cleavage substrate for PPV protease. The construct also contains the HA tag on the C-terminus. The P5 coiled coil dimerizes with the AP6 and P6 coiled coils. If its partner coiled coil (AP6 or P6) contains the N-terminal part of the split luciferase, then dimerization of  the two coils enables luciferase reconstitution due to the close proximity of the split luciferase parts. However, because the linker contains PPV protease cleavage substrate, the cLuc can be cleaved off in the presence of the PPV protease, preventing reconstitution of the split luciferase. </p>
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<p>This construct consists of a C-terminal part of the split luciferase that is fused to the P3 coiled coil (<ref>1</ref>) via a linker which contains the cleavage substrate for PPV protease. The construct also contains the HA tag on the C-terminus. The P3 coiled coil dimerizes with the AP4 and P4 coiled coils. If its partner coiled coil (AP4 or P4) contains the N-terminal part of the split luciferase, then dimerization of  the two coils enables luciferase reconstitution due to the close proximity of the split luciferase parts. However, because the linker contains PPV protease cleavage substrate,   the cLuc can be cleaved off in the presence of the PPV protease, preventing reconstitution of the split luciferase (<ref>2</ref>). </p>
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          <figcaption><b> Sequence alignment of different coils used to tune the affinity of antiparallel coiled-coils.</b><br/> 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).</figcaption>
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          <figcaption><b> Introduction of protease cleavage site between the reporter (effector) and coiled-coil segment(s) </b><br/> Cleavage sites in between CCs and reporter protein introduces logical negation. </figcaption>
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Latest revision as of 18:05, 18 October 2016


P3:GS6:PPVs:GS6:cLuc:HA

This construct consists of a C-terminal part of the split luciferase that is fused to the P3 coiled coil (1) via a linker which contains the cleavage substrate for PPV protease. The construct also contains the HA tag on the C-terminus. The P3 coiled coil dimerizes with the AP4 and P4 coiled coils. If its partner coiled coil (AP4 or P4) contains the N-terminal part of the split luciferase, then dimerization of the two coils enables luciferase reconstitution due to the close proximity of the split luciferase parts. However, because the linker contains PPV protease cleavage substrate, the cLuc can be cleaved off in the presence of the PPV protease, preventing reconstitution of the split luciferase (2).

Sequence alignment of different coils used to tune the affinity of antiparallel coiled-coils.
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).
Introduction of protease cleavage site between the reporter (effector) and coiled-coil segment(s)
Cleavage sites in between CCs and reporter protein introduces logical negation.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 139
  • 1000
    COMPATIBLE WITH RFC[1000]