Difference between revisions of "Part:BBa K3396010"
 
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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.
 
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.
  
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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.
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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.
  
  
 
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<img src="https://static.igem.org/mediawiki/parts/6/61/TRIM21-FRB-P2A-FKBP-GFPnano_Figure1.jpg" alt="" width="360" height="300" />
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<img src="https://static.igem.org/mediawiki/parts/6/61/TRIM21-FRB-P2A-FKBP-GFPnano_Figure1.jpg" alt="" width="250" />
 
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===Method===
 
===Method===
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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.
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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===
 
===Results===
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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).
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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).
  
  
 
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<p><img src="https://2020.igem.org/wiki/images/f/fc/T--NUDT_CHINA--results1105.png" alt="" width="500" /></p>
 
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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.
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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.
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1697
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2581
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1654
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 721