Difference between revisions of "Part:BBa K3396010"

Revision as of 13:48, 25 October 2020

GFPnano-FKBP-P2A-Trim21-FRB

CMV->Kozak->Truncated TRIM21->FRB->P2A->FKBP->GFPnano->BGH-pA

This part is designed to demonstrate a small molecule induced target protein degradation with the Predator Pro system. Herein, FRB-FKBP pairs were used to mediate the rapamycin induced trimerization of truncated Trim21, GFP nanobody and target protein EGFP.

Usage and Biology

Our composite part BBa_K3396010 demonstrates how the Predator Pro system works. It regulates the target protein (EGFP) degradation under the control of external rapamycin signal. As a functional improvement of the existing Part (BBa_K2653016), which degrades EGFP constitutively, this new part provides new interface for external signal control.

The PRYSPRY-lgG Fc interaction of BBa_K2653016 is replaced with FRB-FKBP interaction to realize controlling the beginning and the speed of the degradation. FRB and FKBP are proteins which bind with each other induced by rapamycin. By administrating rapamycin, FRB-FKBP dimerization would trigger the formation of EGFP-GFPnano-trunctaed_Trim21 trimer, in which the truncated Trim21 would mediate the ubiquitylation and degradation of EGFP protein.

Figure 1. Schematic representation of different approaches for this part.

Characterization

This composite part can achieve ubiquitination of target protein. To verify whether it worked, we did a test of it.

Method

Both BBa_K3396010 containing plasmid and EGFP expressing plasmid were transfected into 293T cells as experimental group. While the cells in control group were transfected with EGFP expressing plasmid and empty plasmid. Cells were cultured for 12 h before administrating ?? mM Rapamycin. Fluorescent imaging was performed 24 h post rapamycin stimulation to quantify the fluorescent intensity.

Results

To test whether the GFP levels can be tuned and continuously regulated with rapamycin, co-transfection of a EGFP expressing plasmid and Predator Pro system were performed. It is shown that there was a significantly decrease in GFP fluorescence (Fig 2A). As is shown in the results, GFP was significantly degraded to about 13% of the original level with the appearance of GFP-nano and HA-Trim, which confirmed that GFP Predator could be used to degrade target protein with high efficiency (Figure 2B). Furthermore, it can be observed that as the concentration of rapamycin increases, the degradation effect becomes more obvious(Figure 2C).

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. (B)Intensity quantification of the GFP-Predator Pro transfected group and its negative control transfected with an empty vector pcDNA3.1. HEK293T cells in each group was transfected with GFP-expression plasmid. (C)Heatmap of rapamycin concentration on the degradation effect of GFP-Predator Pro.

Sequence and Features