Difference between revisions of "Part:BBa K3396009"

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<partinfo>BBa_K3396009 short</partinfo>
 
<partinfo>BBa_K3396009 short</partinfo>
  
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CMV->Kozak->Truncated TRIM21->DocS->P2A->Coh2->GFPnano->BGH-pA
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CMV->Kozak->GFPnano->Coh2->P2A->Truncated TRIM21->DocS->BGH-pA
  
 
This part is designed to prove that our Trim21 truncation strategy works as we expected. Since the original Trim-away system utilizes the Trim21 PRYSPRY domain to interact with the antibody IgG, we herein changed such interaction pair into other constitutively dimerization pairs to validate our design.
 
This part is designed to prove that our Trim21 truncation strategy works as we expected. Since the original Trim-away system utilizes the Trim21 PRYSPRY domain to interact with the antibody IgG, we herein changed such interaction pair into other constitutively dimerization pairs to validate our design.
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We expect such system to work in a similar manner as the previous GFP predator as we reported in iGEM 2018.
 
We expect such system to work in a similar manner as the previous GFP predator as we reported in iGEM 2018.
  
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https://2020.igem.org/wiki/images/d/d0/T--NUDT_CHINA--GFPnano-DocS-P2A-Coh2-Trim21_Figure1.png
 
  
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<img src="https://2020.igem.org/wiki/images/d/d0/T--NUDT_CHINA--GFPnano-DocS-P2A-Coh2-Trim21_Figure1.png" alt="" width="360" height="300" />
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</p>
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Figure 1. Schematic representation of different approaches for this part.
 
Figure 1. Schematic representation of different approaches for this part.
  
 
===Characterization===
 
===Characterization===
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This composite part can achieve ubiquitination of target protein. To verify whether it worked, we did a test of it.
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This composite part can achieve ubiquitination of target protein. To verify whether it worked, following tests were conducted.
  
 
===Methods===
 
===Methods===
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===Results===
 
===Results===
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GFP expressing plasmid, and BBa_K3396009 expressing plasmids were co-transfected to HEK-293T cells. 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.
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GFP expressing plasmid, and BBa_K3396009 expressing plasmids were co-transfected to HEK-293T cells. 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, 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 protein degradation activity of this part.
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<p><img src="https://static.igem.org/mediawiki/parts/d/d9/T--NUDT_CHINA--BBa_K3396009-2_Fig1.png" alt="" width="700" height="193" /></p>
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https://2020.igem.org/wiki/images/0/06/T--NUDT_CHINA--GFPnano-DocS-P2A-Coh2-Trim21_Figure2.png
 
 
Figure 2. (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.
 
Figure 2. (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.
  

Latest revision as of 18:01, 27 October 2020


GFPnano-DocS-P2A-Trim21-Coh2

CMV->Kozak->GFPnano->Coh2->P2A->Truncated TRIM21->DocS->BGH-pA

This part is designed to prove that our Trim21 truncation strategy works as we expected. Since the original Trim-away system utilizes the Trim21 PRYSPRY domain to interact with the antibody IgG, we herein changed such interaction pair into other constitutively dimerization pairs to validate our design.


Usage and Biology

The PRYSPRY-lgG Fc interaction of natural Trim21-antibody complex is replaced with DocS-Coh2 to demonstrate that the Trim-Away alike method could still work. DocS and Coh2 are a pair of proteins that has been widely used in synthetic biology to test split protein approach due to their high constitutively dimerization affinity. Theoretically, the dimerization of DocS and Coh2 will bring the truncated Trim21 protein and the target protein EGFP into proximity and form a s stable trimer. Truncated Trim21 would then function as the E3 ligase to ubiquitylate EGFP and mediate its degradation. We expect such system to work in a similar manner as the previous GFP predator as we reported in iGEM 2018.


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, following tests were conducted.

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

GFP expressing plasmid, and BBa_K3396009 expressing plasmids were co-transfected to HEK-293T cells. 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, 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 protein degradation activity of this part.



Figure 2. (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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1790
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1747
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 721