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COMPOSITE PARTS

Favorite Composite Part

TRIM21-Replaceable-1-P2A-Replaceable-2-Replaceable-3(Part: BBa_K3396017)

Our favorite composite part (Part: BBa_K3396017)is a plasmid platform on which different targeting domains and protein dimerization pairs can be easily installed into the Predator Pro system. The subpart (Part: BBa_K3396007)functions as the E3 ligase to degrade target protein. The protein region (Part: BBa_K3396013) and (Part: BBa_K3396014)can be replaced into different protein dimerization pairs in order to achieve signal responsiveness. The (Part: BBa_K3396015) 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 BsaI, BsmBI 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).[The data cannot prove the claim]

Figure 2. (A)Fluorescence images of the GFP-Predator Pro transfected group and its negative control transfected with an empty vector. HEK293T cells in both groups were transfected with GFP-expression plasmid and treated with rapamycin 12 h post transfection. (B)Intensity quantification of the fluorescent images obtained in each well. At least three random sites in each well were picked for imaging to obtain fluorescent intensity. Error bar shows at least three biological replicates.

Links

BBa_K3396002	TRIM21-FRB
BBa_K3396003	FKBP-GFPnano
BBa_K3396004	FKBP-FKBP-GFPnano
BBa_K3396005	Trim21-DocS
BBa_K3396016	CMV-replaceable-Fluc-P2A-Rluc
BBa_K3396009	GFPnano-DocS-P2A-Trim21-Coh2
BBa_K3396010	TRIM21-FRB-P2A-FKBP-GFPnano
BBa_K3396011	GFP-Fluc
BBa_K3396017	TRIM21-Replaceable-3-P2A-Replaceable-2-Replaceable-1
BBa_K3396018	GFPnano-FKBP-FKBP-P2A-Trim21-FRB