Difference between revisions of "Part:BBa K1965047"

 
 
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<partinfo>BBa_K1965047 short</partinfo>
 
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<p>The construct consists of the C-terminal part of the split luciferase (cLuc), adjacent to the coiled coil A which is further connected to its partner coiled coil B'2a via a linker consisting of the PPV protease substrate sequence. The construct also contains the HA tag on the C-terminus. The sequences for the A and B'2a coiled coils were described in Shekhawat <i>et al.</i> (<ref>1</ref>)<sup>[1]</sup>.  </p>
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<p>The A coiled coil with its adjacent cLuc part, dimerizes with its partner coiled coil B that contains the nLuc part. When the two coils dimerize the two parts of the split luciferase come into close proximity, thus regaining their luciferase activity. However, dimerization of the two chains is prevented by the presence of an antiparallel coiled coil segment B’2a that inhibits the binding of its partner to other coiled coil peptides. Reconstitution is enabled by the proteolytic cleavage of the linker between the coiled coil, fused to the split reporter and the autoinhibitory segment. The latter dissociates from the complex and can therefore be replaced by the coiled-coil forming peptide with the second segment of the split reporter. This construct was combined with the construct (<a href="https://parts.igem.org/Part:BBa_K1965048">BBa_K1965048</a>), which contained the TEV protease cleavage substrate between two coils, to design AND logic gates. The addition of PPV and TEV proteases thus enabled both luciferase splits to come into close proximity and regain their catalytic activity (<ref>2</ref>B).  </p>
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          <figcaption><b> Sequence alignment of different coiled 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> Interactions and protease activated AB coiled-coil formation.</b><br/> 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. </figcaption>
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<sup>[1]</sup> Shekhawat, S. S., Porter, J. R., Sriprasad, A., & Ghosh, I. (2009). An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease Sensors. Jacs, 131(42), 15284–15290. <br>
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Latest revision as of 17:38, 18 October 2016


cLuc:A:PPVs:B'2a:HA

The construct consists of the C-terminal part of the split luciferase (cLuc), adjacent to the coiled coil A which is further connected to its partner coiled coil B'2a via a linker consisting of the PPV protease substrate sequence. The construct also contains the HA tag on the C-terminus. The sequences for the A and B'2a coiled coils were described in Shekhawat et al. (1)[1].

The A coiled coil with its adjacent cLuc part, dimerizes with its partner coiled coil B that contains the nLuc part. When the two coils dimerize the two parts of the split luciferase come into close proximity, thus regaining their luciferase activity. However, dimerization of the two chains is prevented by the presence of an antiparallel coiled coil segment B’2a that inhibits the binding of its partner to other coiled coil peptides. Reconstitution is enabled by the proteolytic cleavage of the linker between the coiled coil, fused to the split reporter and the autoinhibitory segment. The latter dissociates from the complex and can therefore be replaced by the coiled-coil forming peptide with the second segment of the split reporter. This construct was combined with the construct (BBa_K1965048), which contained the TEV protease cleavage substrate between two coils, to design AND logic gates. The addition of PPV and TEV proteases thus enabled both luciferase splits to come into close proximity and regain their catalytic activity (2B).

Sequence alignment of different coiled 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).
Interactions and protease activated AB coiled-coil formation.
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.
[1] Shekhawat, S. S., Porter, J. R., Sriprasad, A., & Ghosh, I. (2009). An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease Sensors. Jacs, 131(42), 15284–15290.



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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]