Part:BBa_K4016031
2x Rb CHelix-CIB1
This compensate part is designed to generate cyclin D degradation with BBa_K4016039 through CIB1-CRY2 interaction.
Usage and Biology
Our project this year is centered on the degradation of proteins by blue light. Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact with CIB1 (Part:BBa K2980002). Functionally, it is used to fuse with other protein and bring them together into phase under light. This pair’s combination under blue light is used as our “On System”.
We focused on degrading Cyclins (mainly CyclinD,E) this year. The mammalian cell cycle is driven by a complex of cyclins and their associated cyclin-dependent kinases (CDKs). Abnormal dysregulation of cyclin-CDK is a hallmark of cancer.[1] We hope our system can contribute to the treatment of diseases like superficial cancer which related to abnormal content of Cyclins.
A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation.[2] Rb phosphorylation promotes its dissociation from E2Fs and thereby drives the expression of E2F-target genes that initiate DNA replication.[3] As cells progress through G1, cyclin D-Cdk4,6 gradually phosphorylates Rb and triggers the onset of E2F-dependent expression of cyclins E and A, Cyclins E and A then bind Cdk1 and Cdk2 to form complexes that continue to phosphorylate Rb. Research shows the cyclin D-Cdk4,6 phosphorylates and inhibits Rb via a C-terminal helix and that this interaction is a major driver of cell proliferation. Additionally, it is Cyclin D, but not other cyclins (Cyclin A/B/E/G/H), targets a C-terminal alpha-helix docking motif on Rb. So, it is reasonable that we use Rb αCHelix instead of its other parts to achieve the recruit of Cyclin D- Cdk4,6.
In summary, the Rb αCHelix is linked with CIB1 in this compensate part, and the CIB1 is linked with Trim21(see BBa_K4016039). Rb αCHelix combines Cyclin D and CIB1 combines Cry2 under the blue light, then Trim21 take the system to UPS for degradation. We hope this part can contribute to the treatment of Cyclin D-CDKs-related diseases.
Special design
We used Rb αCHelix instead of other parts of Rb or the whole Rb in this part. That’s because Rb αCHelix specifically combines with Cyclin D-CDKs, but not with other Cyclins. By doing this we can ensure that we have indeed degraded, and only just degraded CyclinD.
- Here is the mechanism of the recombined 2x Rb αCHelix-CIB1:
1.The Rb αCHelix tagged with CIB1 combines with Cyclin D-CDKs.
2. TRIM21-CRY2 (see BBa_K4016039) connect CIB1- Rb αCHelix -target through the CRY2-CIB1 interaction.
3. The targeted protein 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’-TGCCACCATGTCTAAATTCCA-3’
R-Prime: 5’-AGCTCGAGAAGACCGACTACATC-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]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 313
- 1000COMPATIBLE WITH RFC[1000]
Functional test
This composite part was constructed with Part:BBa_K4016035. Thus, the function of this composite part was validated together with Part:BBa_K4016035.
Method
- 1. Cell transfection
(1)Seed HEK293T cells into 6-well cell culture plates.
(2)Culture for 16 h before transfection
(3)Total plasmid mixes of 800ng per well are mixed thoroughly in DMEM before a polyethylenimine (PEI) solution (1 mg/ml) is added into the plasmid mixture in a ratio of 1:5 (plasmid weight/PEI weight)
(4)The plasmid–PEI mixture is vortexed and incubated at room temperature for 15 min. The mixture is then added into the cells and incubated for at least 6 h.
(5)Cells are then changed into fresh medium and culture for 18 h before subculture.
- 2.CCK-8 assay
(1)Wash HEK293T cells in 6-well plate with PBS and trypsinize prior to resuspension in fresh complete medium in a 15 ml microcentrifuge tube.
(2)Dispense 100ul of cell suspension (approximately 30000 cells per well) into 96 well plates.
(3)Apply the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48/72 h before sampling and analysis assay
(4)Add 10 ul CCK-8 solution to each well and incubate for 2 h in the incubator.
(5)Record results using microplate reader to measure the absorbance at 450 nm.
Figure2. Experimental validation approach
Result
To demonstrate 2 x Rb CHelix-CIB1 can interact with Trim21-Cry2 when stimulated with blue light and then result in Cyclin D degradation，we did CCK-8 assay.
The result showed a significant decrease of 450nm absorbance compared to the control group (transfected with pcDNA3.1), indacating that in the experimental group, the growth of cells was inhibited. The result successfully proved our system can work as we expected.
Reference
[1]Gao X , Leone G W , Wang H . Cyclin D-CDK4/6 functions in cancer[J]. Advances in Cancer Research, 2020, 148.
[2]Cyclin D-Cdk4,6 drives cell cycle progression via the retinoblastoma protein's C-terminal helix. 2018. Bertoli C , Skotheim J M , RAMD Bruin. Control of cell cycle transcription during G1 and S phases[J]. Nature Reviews Molecular Cell Biology, 2013, 14(8):518.
[3]Bertoli C , Skotheim J M , RAMD Bruin. Control of cell cycle transcription during G1 and S phases[J]. Nature Reviews Molecular Cell Biology, 2013, 14(8):518.
| None |