Part:BBa_K4016035
Trim21-CRY2
This composite part is designed to generate GFP degradation with GFPnano-CIB1(Part:BBa_K4016036) through CRY2-CIB1 dimerization[3].
Usage and Biology
This part is composed of Cryptochrome 2(CRY2) photoreceptor linked to Trim21[4][5]. When induced by blue light, CRY2 dimerizes with its binding partner CIB1, effectively bringing the target site defined antibody GFP-nano[1][2]. While achieving the purpose of optical control, Trim21 bind with antibody GFP-nano to prove that the PREDATOR PRO really works.
Figure 1. Schematic figure of BBa_K4016035 and BBa_K4016036
- Here is the mechanism of the recombined Trim21-CRY2:
1.Trim21-CRY2 connect with GFPnano-CIB1 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’-TGCTGGATATCTGCAGAATTCttaGGGAGCGGCGCCGATCAT-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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 204
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1195
Illegal BglII site found at 1654
Illegal BamHI site found at 2133 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 161
Illegal AgeI site found at 1079
Illegal AgeI site found at 1808 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1431
Illegal BsaI.rc site found at 840
Illegal SapI.rc site found at 948
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.
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