Team:NUDT CHINA/Basic Parts

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BASIC PARTS

Favorite Basic Parts

Truncated Trim21 (Part: BBa_K3396007)

It is the core of the newly registered Predator Pro system. Trim 21 comes from the family of TRIM. We truncated the Trim21 protein to maintain its RING domain, B box domain and coiled coli domain, while the antibody binding PRYSPRY domain was removed. This truncate protein maintained the E3 ubiquitin ligase activity, and provided an open interface for other protein dimerization pairs to be added.

Characterization

We replaced the PRYSPRY structure of truncated trim21 with a DocS-Coh2 protein pair, and attached a GFPnano antibody to the end of Coh2 to achieve specific degradation of the GFP protein. This new part was registered as BBa_K3396009.

Methods

Both the plasmids carrying BBa_K3396009 or EGFP expressing cassette were transfected into HEK-293T cells as experimental group. While the cells in control group were co-transfected GFP expressing plasmid and empty plasmid.

Results

A significant decrease of green fluorescence was observed in GFP Predator transfected groups 48 hours after transfection indicating the decrease of GFP protein level (Figure 2A). Subsequently, the Western blotting analysis was performed to examine the level EGFP protein. As shown in the results, EGFP was significantly degraded to about 30% of the original level with the appearance of BBa_K3396009 (Figure 2B), which confirmed that Predator could be used to degrade target protein with high efficiency.

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.

CMV-Replaceable-1 -Fluc-P2A-Rluc(Part: BBa_K3396016)

Usage and Biology

(Part:BBa_K3396008)is a Dual Luciferase Reporter system to quantify the abundance of specific target protein. Dual luciferase assay was introduced to normalize the differences caused by irrelevant factors.

Special Design

Special designs were taken to optimize the applicability and adaptivity of such parts. Specifically, a novel designed substitution system, through which, target proteins could be fused with Fluc fragment using Golden-Gate Assembly, was introduced to dramatically simplify the cloning process.

Characterization

We use Golden-Gate Assembly to fuse GFP with Fluc fragment. This system can be used to quantify the abundance of specific target protein. We compared the degradation curves of GFP and Fluc as reporter molecules, respectively and result showed that the detection of Fluc is more accurate.

Figure 2.Detection accuracy of different reporter system.

Links

BBa_K3396000	DocS
BBa_K3396001	Coh2
BBa_K3396007	Truncated Trim21
BBa_K3396013	Replaceable-1
BBa_K3396014	Repleceable-2
BBa_K3396015	Repleceable-3

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                     P.R.China