Part:BBa_K3396009

GFPnano-DocS-P2A-Trim21-Coh2

CMV->Kozak->Truncated TRIM21->DocS->P2A->Coh2->GFPnano->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, we did a test of it.

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, 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.

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