Difference between revisions of "Part:BBa K1965051"

 
 
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<partinfo>BBa_K1965051 short</partinfo>
 
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<p>This construct consists of the N-terminal part of the split luciferase that is fused to the AP4 coiled coil (<ref>1</ref>) via a linker which contains the cleavage substrate for TEV protease. The construct also contains the Myc tag on the N-terminus. The AP4 coiled coil dimerizes with the P3 coiled coil. If its partner coiled coil (P3) contains the C-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 TEV protease cleavage substrate,  the nLuc can be cleaved off in the presence of the TEV 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).
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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.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 17:51, 18 October 2016


Myc:nLuc:GS6:TEVs:GS6:AP4

This construct consists of the N-terminal part of the split luciferase that is fused to the AP4 coiled coil (1) via a linker which contains the cleavage substrate for TEV protease. The construct also contains the Myc tag on the N-terminus. The AP4 coiled coil dimerizes with the P3 coiled coil. If its partner coiled coil (P3) contains the C-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 TEV protease cleavage substrate, the nLuc can be cleaved off in the presence of the TEV 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 103
    Illegal NgoMIV site found at 1447
    Illegal NgoMIV site found at 1468
    Illegal AgeI site found at 1171
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1565
    Illegal SapI.rc site found at 1353