Difference between revisions of "Part:BBa K3147010:Design"

Revision as of 16:36, 20 October 2019

Mutant TEV (PE10) protease fused to anti-sfGFP VHH with TEV-cleavable maltose binding protein

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
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 77
Illegal BsaI.rc site found at 2377
Illegal SapI.rc site found at 1868

Design note

The TEV cutting site used is a modified site from the original consensus sequence: ENLYFE/G [3].

The TEV protease used is a mutant TEV with the mutation T17A, T146A, D148P, S153C, S168T, S170A, T173A, S219P [3].

The Terminator used is a double terminator (Part:BBa_0015).

The VHH is a VHH directed against the sfGFP [4], it recognizes a conformational pattern.

Reference

[3] Yi, L. et al. 2013. « Engineering of TEV Protease Variants by Yeast ER Sequestration Screening (YESS) of Combinatorial Libraries ». Proceedings of the National Academy of Sciences 110(18): 7229‑34

[4] Kubala, M. H., Kovtun, O., Alexandrov, K., & Collins, B. M. (2010). Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein science : a publication of the Protein Society, 19(12), 2389–2401. doi:10.1002/pro.519

@@ Line 10: / Line 10: @@
 The TEV protease used is a mutant TEV with the mutation T17A, T146A, D148P, S153C, S168T, S170A, T173A, S219P [3].
-The Terminator used is a double terminator ([[BBa_0015]]).
+The Terminator used is a double terminator ([[Part:BBa_0015]]).
 The VHH is a VHH directed against the sfGFP [4], it recognizes a conformational pattern.