Part:BBa_K4016028
2x CDK4 αCHelix-Coh2
This fusion protein is combined 2x CDK4 αCHelix (Part:BBa_K4016009) with Coh2 (Part:BBa_K3396001).
Usage and Biology
CDK4 αCHelix is the αCHelix of CDK4, a member of growth-dependent cyclin-dependent kinase (CDK) family. Interacting with Rb αCHelix (Part:BBa_K4016010), the activation of CDK4 is a key response to growth factors in many cell types.[1]
The Coh2 module comes from C. thermocellum and it could recognize and bind tightly to complementary DocS modules harbored by each of the catalytic subunits. [2]The Coh2–DocS pair represents the interaction between two complementary families of protein modules that exhibit divergent specificities and affinities, ranging from one of the highest known affinity constants between two proteins to relatively low-affinity interactions.
By combining these two domains, we can achieve precisely inhibiting the retinoblastoma protein leading to G1–S cell-cycle transition by the specific binding of Docs and Coh2.
Special design
Considering other structures may disrupt the position of the specific helix[1] or produce other impacts, the CDK4 is truncated to maintain its αCHelix to exclude the effects of other factors as far as possible.
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’-GGGAGCGGAACAGGCTCCGGGACCGGAAGCGTGGTGGTGGAGATCGGCAAG-3’
R-Prime: 5’-TGGATATCTGCAGAATTCTTACACGTTCACGCCGCCGTCGAT-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]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Functional test
We used CCK-8 to observe the cell proliferation, and further evaluate the degradation effect of our system.
Method
1.Inoculate cells in a cell culture flask or dish, and allow cells to adhere or grow for approximately 4–24 hours before proceeding with the assay.
2.Add 110 volume of Cell Counting Kit-8 (CCK-8) directly to cells in culture medium. Mix thoroughly to achieve a homogenous solution by lightly tapping the outside of the plate several times while avoiding bubbles. For 96-well plate, add 10 µl Cell Counting Kit-8 (CCK-8) per 100 µl culture medium.
3.Incubate in a cell culture incubator for 0.5 to 4 hours at 37°C until the color turns orange. Over incubation will give false results.
4.Record results using microplate reader to measure the absorbance of Cell Counting Kit-8 (CCK-8) at 450 nm. Recommended OD values range between 0.8-1.5, however values between 0.5-2.5 are acceptable.
5.Optional: Add 10 µl of 1 % SDS (dissolve 0.1 g SDS with PBS buffer to prepare 10 ml solution) directly to 100 µl of cells to stop the reaction. Signals can be read within 3 days without affecting the absorbance values.
6.Calculation of cell viability: Cell viability (%) = [A (Drug+) – A (Black)] [A (Drug-) – A (Black)] x 100% A (Drug+) : OD value of wells with cells, CCK-8 and drugs; A (Drug-) : OD value of wells with cells, CCK-8, but without drugs; A (Black): OD value of wells with culture medium and CCK-8, but without cells.
Result
To validate our system can apply to practical situations, we designed part 2x CDK4 αCHelix-Coh2 (Part:BBa_K4016028), which can target to CyclinD thus regulating the cell cycle as well as interact with Docs. The result showed a significant decrease of 450nm absorbance compared to the control group, indacating that in the experimental group, the growth of cells was inhibited. The result successfully proved our system can work as we expected—target and degrade CyclinD, therefore apply to some trearments and other fields.
Reference
[1] Morgan DO. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol. 1997;13:261-291. doi:10.1146annurev.cellbio.13.1.261
[2] Barak, Y. et al. Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction. Journal of Molecular Recognition 18, 491-501, doi:10.1002jmr.749 (2005)
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