Part:BBa_K4016035
Trim21-CRY2
This composite part is designed to generate GFP degradation with GFPnano-CIB1(Part:BBa_K4016036) through CRY2-CIB1 dimerization.
Usage and Biology
This part is composed of Cryptochrome 2(CRY2) photoreceptor linked to Trim21. When induced by blue light, CRY2 dimerizes with its binding partner CIB1, effectively bringing the target site defined antibody GFP-nano. 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
Functional test
Method
- Double luciferase reporter gene detection
1.Preparation: Abandon the supernatant. Use PBS to wash cells. Abandon the added PBS.[two lines after two lines; separate to two steps]
2.Lytic cells: after fully mixing the reporter gene cell lysate, add the reporter gene cell lysate to fully lyse the cells. After absorbing the cell culture medium, add reporter gene cell lysate with 200uL / hole in 24 orifice plate. Place the culture plate on a rocking platform or orbital shaker with gentle motion .
3.After full cracking, cryopreservation(N2;liquid).Then frozen samples need to be tested at room temperature.
4.14000g centrifuge for 10min. Then suck up the supernatant for the test later.
5.The firefly luciferase detection reagent and the sea kidney luciferase detection buffer were melted and reached room temperature. The sea kidney luciferase detection substrate (100x) is placed on an ice bath or ice box.
6.According to the amount of 50 uL needed for each sample, take appropriate amount of sea kidney luciferase detection buffer and add sea kidney luciferase detection substrate (100x) to prepare sea kidney luciferase detection working solution(1:100).
7.Turn on the chemiluminescence meter or the multi-function enzyme labeling instrument with the function of detecting chemiluminescence according to the operating instructions of the instrument. the determination interval is set to 2 seconds and the determination time is set to 10 seconds. (each 12 seconds plus reagent; test in 6min)
8.When determining each sample, take 30uL.The usage of the same batch of samples should be consistent.
9.Add 50uL of firefly luciferase detection reagent, beat well with a gun or mix well in other appropriate ways, and then determine RLU (relativelightunit).
10.After completing the above steps for the determination of firefly luciferase, add 50uL of sea kidney luciferase detection solution, beat it well with a gun or mix it in other appropriate ways, and then determine RLU (relativelightunit).
11.When the sea kidney luciferase was used as the internal reference, the LUR value determined by firefly luciferase was divided by that determined by sea kidney luciferase. According to the obtained ratio, the activation degree of reporter gene among different samples was compared. If firefly luciferase is used as the internal reference, similar calculation can also be carried out.
Result
Figure 2. The result of double luciferase reporter gene detection
To prove PREDATOR PRO really works, we use the experiment--double luciferase reporter gene detection to verify its outstanding ability of protein degradation under blue light inducing. The result showed that on the condition of blue light inducing, the efficiency of protein degradation is significantly better than in the dark condition, which specifically shows the PREDATOR PRO is workable.
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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