Part:BBa_K3396017
Replaceable1-Replaceable2-P2A-Truncated Trim21-Replaceable3
CMV->Replaceable1->linker->Replaceable2->P2A->Truncated Trim21->Replaceable3->BGH
This part is a composite part providing Golden Gate assembly based rapid construction of PREDATOR Pro plasmids for controlled degradation of specific target protein.
Usage and Biology
Our favorite composite part BBa_K3396012 is a plasmid platform on which different targeting domains and protein dimerization pairs can be easily installed into the Predator Pro system. The subpart BBa_K3396007 functions as the E3 ligase to degrade target protein. The protein region BBa_K3396014 and BBa_K3396015 can be replaced into different protein dimerization pairs in order to achieve signal responsiveness. The BBa_K3396013 part can be replaced by other targeting domains (ScFv. Nanobody, ligand, etc.) to mediate targeted degradation. The parts BBa_K3396013- BBa_K3396015 were designed with flanking BsmBI, BsaI and AlwI sites for the scarless replacement of these parts.
Special Design
As a member of the collection Predator system, special designs were taken for to optimize the applicability and adaptivity of such parts. Specifically, a novel designed substitution system, through which, two proteins could be fused with their corresponding fragment at the same time using Golden-Gate Assembly, was introduced to dramatically simplify the cloning process.
Characterization
We use Golden-Gate Assembly to replace Replaceable-1 with FRB fragment, Replaceable-2 with FKBP-FKBP fragment, Replaceable-3 with GFPnano fragment. This system can be used to degrade the specific GFP. This new part is registered as BBa_K3396010. To test whether the GFP levels can be tuned and continuously regulated by rapamycin, co-transfection of a EGFP expressing plasmid and Predator Pro system were performed. Result showed that there was a significantly decrease in GFP fluorescence (Fig 2A) in BBa_K3396010 expressing group comparing to the control group. Florescence quantification showed that GFP was significantly degraded to about 13% of the original level with the appearance of BBa_K3396010, which confirmed that GFP Predator could be used to degrade target protein with high efficiency (Figure 2B).
Figure 1.(A) Fluorescence images and intensity quantification of BBa_K3396009 transfected group and its negative control group. HEK-293T cells in both groups were transfected with GFP-expression plasmid. (B) Western blotting determining the expression level of GFP in HEK-293T cells transfected with BBa_K3396009.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1106
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1063
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 840
Illegal BsaI site found at 1789
Illegal BsaI.rc site found at 786
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