Original parts

Functional unit of the PR PREDATOR, expressing fusional GFPnano-hIgG1-FC and HA tag-Trim21 by automatic cleavage of P2A. (Part: BBa_K2653016)

Our improvement

Our improved part is a Rapamycin-induced Predator Pro system (Part: BBa_K3396010) in which we replaced the PRYSPRY/Fc domain in original part with FRB-FKBP domain. Since the previous part achieved degradation effect in transcriptional level，we hope that our improved part could manipulate protein degradation in a more direct, accurate, acute way. This year we have made a series of modification based on the Minimum TATA-box promoter designed by Jiaxin Ma Group of NUDT_CHINA. (BBa_K2653016).In order to achieve a signal control of target protein degradation, we replaced the Trim21 PRYSPRY domain and its interacting partner IgG-Fc domain into the FRB and FKBP, a set of widely used and fully characterized heterodimerizing components regulated 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.

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 12h before administrating 2ng/ul mM Rapamycin. Fluorescent imaging was performed 24h 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).