Difference between revisions of "Part:BBa K3396010"

Latest revision as of 03:24, 28 October 2020

GFPnano-FKBP-P2A-Trim21-FRB

CMV->Kozak->GFPnano->FKBP->P2A->Truncated TRIM21->FRB->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 of both initiation and rate of the degradation. FRB and FKBP are proteins which bind with each other under induction of 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 Rapamycin. Fluorescent imaging was performed 48 h post rapamycin stimulation to quantify the fluorescent intensity.

Results

To test whether the GFP levels can be tuned and continuously regulated by rapamycin, HEK-293T cells were co-transfected with GFP expressing plasmid and RiPrePro1.0 plasmid/empty vector. Fluorescent imaging showed slight decrease (~20%) of GFP fluorescence in RiPrePro1.0 expressing group comparing to the control group (Fig 2A and 2B) under 1 ng/μL rapamycin induction. Also, increasing concentration of rapamycin significantly improve degradation effect (Fig 2C).

Figure 2. (A)Representative fluorescence images of the RiPrePro-1 transfected group and negative control group transfected with an empty vector. HEK293T cells in both groups were co-transfected with GFP-expression plasmid. Cells were induced with 1 ng/μL rapamycin 24 h post transfection. Images were taken 48 h post induction. (B) Quantification of fluorescent intensity in (A) and its parallel biological replicates. (C)Heatmap of GFP Fluorescent intensity of BBa K3396010 carrying palsmid or empty vector transfected, GFP expressing HEK-293T cells treated with gradient rapamycin concentration.

Sequence and Features