Difference between revisions of "Part:BBa K3396007"
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BBa_K3396007 is the core of the newly registered Predator Pro system. Trim 21 comes from the family of TRIM. We truncated the Trim21 protein to maintain its RING domain, B box domain and coiled coli domain, while the antibody binding PRYSPRY domain was removed. This truncate protein maintained the E3 ubiquitin ligase activity, and provided an open interface for other protein dimerization pairs to be added.  
 
BBa_K3396007 is the core of the newly registered Predator Pro system. Trim 21 comes from the family of TRIM. We truncated the Trim21 protein to maintain its RING domain, B box domain and coiled coli domain, while the antibody binding PRYSPRY domain was removed. This truncate protein maintained the E3 ubiquitin ligase activity, and provided an open interface for other protein dimerization pairs to be added.  
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===Characterization===
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We replaced the PRYSPRY structure of truncated trim21 with a DocS-Coh2 protein pair, and attached a GFPnano antibody to the end of Coh2 to achieve specific degradation of the GFP protein. This new part was registered as BBa_K3396009.
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===Method===
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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.
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===Results===
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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 1A). 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 1B), which confirmed that Predator could be used to degrade target protein with high efficiency.
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<p><img src="https://static.igem.org/mediawiki/parts/0/0d/T--NUDT_CHINA--truncated_trim21_Fig1.png" alt="" width="500" /></p>
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</html>
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Figure 1. (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.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3396007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3396007 SequenceAndFeatures</partinfo>

Revision as of 16:15, 27 October 2020


Truncated Trim21

This part is the truncated version of human Trim21 coding sequence. We removed the PRYSPRY domain containing 268-475 aa of the original Trim21 to allow further engineering.

Usage and Biology

BBa_K3396007 is the core of the newly registered Predator Pro system. Trim 21 comes from the family of TRIM. We truncated the Trim21 protein to maintain its RING domain, B box domain and coiled coli domain, while the antibody binding PRYSPRY domain was removed. This truncate protein maintained the E3 ubiquitin ligase activity, and provided an open interface for other protein dimerization pairs to be added.

Characterization

We replaced the PRYSPRY structure of truncated trim21 with a DocS-Coh2 protein pair, and attached a GFPnano antibody to the end of Coh2 to achieve specific degradation of the GFP protein. This new part was registered as BBa_K3396009.

Method

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

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 1A). 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 1B), which confirmed that Predator could be used to degrade target protein with high efficiency.



Figure 1. (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 204
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 161
  • 1000
    COMPATIBLE WITH RFC[1000]