Team:NUDT CHINA/Composite Parts
COMPOSITE PARTS
2×Rb CHelix-CIB1 (BBa_K4016031)
Since optogenetic tools have been reported to play important roles in cell signaling pathway regulation, using optogenetic tools in cells have been developed rapidly for years. On the basis of the Predator Pro system designed by our team in 2020, we replaced our core module with a module controlled by blue light, and our targeting module was replaced with Cyclins. In our registered and submitted parts, we provide three blue-light-mediated modules(Gry2-CIB1,PixD-PixE,lov2-zdk), and many parts to connect with cyclins. Our favorite composite part 2×Rb CHelix-CIB1(BBa_K4016031) is constructed with the basic part Rb αChelix(BBa_K4016010), and CIB1(BBa_K2980002) (Figure 1).
Figure 1. Schematic representation of the Rb αHelix’s function on targeting cyclinD1 in the CycleBlue System.
Actually, the composite part "2×Rb CHelix-Coh2"(BBa_K4016032) is a prototype of this part. First, through interaction between composite parts "Trim21-DocS"(BBa_K3396005) and "2×Rb CHelix-Coh2"(BBa_K4016032), we can verify that 2×Rb CHelix can be combined with Cyclin D-CDKs as a targeting module, thus confirming the effectiveness of them(Figure 2C). Further more, to improve our system,we turned the core module into parts CRY2 and CIB1(BBa_K2980000 and BBa_K2980002) that can be mediated by blue light.
Figure 2. 2×Rb CHelix-Coh2 related experiments and experimental results. (A) Schematic representation of the experimental process of 2×Rb CHelix-CIB1 targeting function validation. (B) Representative microscopic images of HEK-293T cells transfected with indicated plasmids. (C) CCK8 cell proliferation assay showing the 450nm Absorbance of HEK-293T cells transfected with pcDNA3.1(control group) and Trim21-DocS+2×Rb CHelix-Coh2(experimental group). This test takes 20min as the interval time. For (C), data represent mean ± s.e.m. (n=3 biological replicates); ***P ＜ 0.001; two-tailed unpaired Student’s t-test.
Cyclin D1 plays a critical role in cell cycleregulation by promoting G1-S phase transition through activation of cyclin-dependent kinase CDK4/6. 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. That’s why we use Rb αCHelix instead of other parts of Rb or the whole Rb in this part to target Cyclin D only1. At the same time, retaining the smallest effective binding domain “αCHelix” can also minimize the interference of Rb as a signal to the cell cycle.
Figure 3. Schematic of the cyclin-Cdk docking sites and the accessible Cdk phosphorylation sites on Rb.
To proof the function of this composite part, we used Cell Counting Kit-8 (CCK-8) through sensitive colorimetric assays to determinate cell viability in cell proliferation. Through the induction of blue light, CIB and CRY2 will combine and cyclinD-CDK4,6 will be degraded by ubiquitin-proteasome system recruited by Trim21. Transfect our target plasmids into HEK-293T cells and dispense 100μl of cell suspension (3000 cells/well) into a 96-well plate. Apply the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48/72 hours in a incubator (at 37°C, 5% CO2). In 24th/48th/72th hour, dilute 10 µL CCK-8 solution to 100 µL, add 100μl diluted CCK-8 solution to each well of the plate. Finally, measure the absorbance at 450 nm using a microplate reader.
Value of OD450 showed significant variation between the blue light positive group and the blue light negative group. Thus then validated the function of this part.
| 01 | BBa_K4016012 | VH_CyclinE1-Coh2 | 02 | BBa_K4016013 | VK_CyclinE1-Trim21-DocS | 03 | BBa_K4016014 | ScFv_CyclinE1-Coh2 |
| 04 | BBa_K4016015 | Coh2-ScFv_CyclinE1 | 05 | BBa_K4016016 | Coh2-VH_CyclinE1 | 06 | BBa_K4016017 | Trim21-DocS-VK_CyclinE1 |
| 07 | BBa_K4016018 | VK_CyclinE1-Docs-Trim21 | 08 | BBa_K4016019 | HA-Trim21-PixE | 09 | BBa_K4016020 | GFPnano-PixD |
| 10 | BBa_K4016021 | HA-Trim21-PixD | 11 | BBa_K4016022 | GFPnano-PixE | 12 | BBa_K4016023 | HA-Trim21-LD3 |
| 13 | BBa_K4016024 | GFPnano-Bcl-xl | 14 | BBa_K4016025 | HA-Trim21-asLOV2 | 15 | BBa_K4016026 | Zdk-GFPnano |
| 16 | BBa_K4016027 | 2×CDK4 αCHelix-CIB1 | 17 | BBa_K4016028 | 2×CDK4 αCHelix-Coh2 | 18 | BBa_K4016029 | VH_CyclinE1-CIB1 |
| 19 | BBa_K4016030 | VK_CyclinE1-Trim21-Cry2 | 20 | BBa_K4016031 | 2×Rb CHelix-CIB1 | 21 | BBa_K4016032 | 2×Rb CHelix-Coh2 |
| 22 | BBa_K4016033 | CIB1-VH_CyclinE1 | 23 | BBa_K4016034 | Trim21-Cry2-VK_CyclinE1 | 24 | BBa_K4016035 | Trim21-CRY2 |
| 25 | BBa_K4016036 | GFPnano-CIB1 | 26 | BBa_K4016037 | ScFv_CyclinE1-CIB1 | 27 | BBa_K4016040 | Trim21-3×GS linker-CRY2 |
| 28 | BBa_K4016041 | GFPnano-3×GS linker-CIB1 | 29 | BBa_K4016042 | Trim21-5×GS linker-CRY2 | 30 | BBa_K4016043 | GFPnano-5×GS linker-CIB1 |
- Topacio, B. R. , Zatulovskiy, E. , Cristea, S. , Xie, S. , Tambo, C. S. , & Rubin, S. M. , et al. (2019). Cyclin d-cdk4,6 drives cell-cycle progression via the retinoblastoma protein's c-terminal helix. Molecular Cell.