Part:BBa_K4016036

GFPnano-CIB1

This composite part is designed to generate GFP degradation with Trim21-CRY2(BBa_K4016035) through CRY2-CIB1 dimerization.

Usage and Biology

This part is composed of antibody GFP-nano by CIB1. When induced by blue light, CIB1 dimerizes with its binding partner CRY2, effectively bringing the target site defined Trim21. While achieving the purpose of optical control, the antibody GFP-nano bind with both Trim21 and the antigen GFP to prove that the PREDATOR PRO really works.

Figure 1. Schematic figure of BBa_K4016036 and BBa_K4016035

• Here is the mechanism of the recombined GFPnano-CIB1:

1.GFPnano-CIB1 connect with Trim21-CRY2 through CRY2-CIB1 interaction and forms a dimerized complex.

2.Inside the complex, GFPnano-CIB1 targets GFP.

3.GFP is degraded by ubiquitin-proteasome system recruited by Trim21

Characterization

This part was validated through four ways:PCR, enzyme digestion, sequencing and functional test.

PCR

The PCR is performed with Green Taq Mix by Vazyme.

F-Prime: 5’-CTAGCGTTTAAACTTAAGCTTggtaccATTTAAATGCCA-3’

R-Prime: 5’-TGCTGGATATCTGCAGAATTCttaGATGTAGTCGGTCTT-3’

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose gel.

Enzyme Digestion

After the assembly ,the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB, we minipreped the plasmid for cutting. The preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from TIANGEN. The cutting procedure was performed with XbaI and KpnI restriction endonuclease bought from TAKARA.

The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.

Sequecing

The plasmid was sequenced correct.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 466
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 35
  

Reference

1.Kennedy, M. J. et al. Rapid blue-light–mediated induction of protein interactions in living cells. Nat Methods 7, 973–975 (2010).

2.Bugaj, L. J., Choksi, A. T., Mesuda, C. K., Kane, R. S. & Schaffer, D. V. Optogenetic protein clustering and signaling activation in mammalian cells. Nat Methods 10, 249–252 (2013).

3.Taslimi, A. et al. Optimized second-generation CRY2–CIB dimerizers and photoactivatable Cre recombinase. Nat Chem Biol 12, 425–430 (2016).

4.Foss, S. et al. TRIM21—From Intracellular Immunity to Therapy. Front. Immunol. 10, 2049 (2019).

5.Liu, C. et al. Predator: A novel method for targeted protein degradation. http://biorxiv.org/lookup/doi/10.1101/2020.07.31.231787 (2020) doi:10.1101/2020.07.31.231787.