Part:BBa_K4016010

2XRb αChelix This part contains two copies of Rb C helix (to ensure the expression of Rb and remove the influence of other parts of Rb on the experiment) and two copies of GS Linker (5xGS Linker & 2xGS Linker). We utilize it to target cyclinD1 through our predator system.

Usage and Biology

Our project this year is centered on the degradation of the cyclins. 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. The Cdk4 and Cdk6 (Cdk4,6) are activated by the D-type cyclins D1, D2, and D3 (cyclin D) to drive cell-cycle progression from G1 to S phase. A body of evidence shows that the Cyclin Ds-CDKs axis plays a critical role in cancer through various aspects, such as control of proliferation, senescence, migration, apoptosis, and angiogenesis.[1]

A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb (Figure 1), 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 (Figure 2).[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 (Figure 3).

Figure1. Schematic of the cyclin-Cdk docking sites and the 14 accessible Cdk phosphorylation sites on Rb.[2]

Figure2. In mammalian cells, the transcriptional repressors RB, p107 and p130 (collectively known as pocket proteins) are bound to E2F transcription factors to repress expression during early G1. Pocket proteins either prevent activator E2F proteins (such as E2F1, E2F2 and E2F3) to activate transcription or function as co repressors for repressor E2F proteins (such as E2F4).

Figure3. Identification of cyclin D-specific helix-based docking.[2]

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 (Figure 4). We intend to use our system to degrade Cyclin Ds-CDKs by its combination of Rb C Helix, to achieve apoptosis. We hope this part can contribute to the treatment of Cyclin Ds-CDKs-related diseases.

Figure 4. This is a model for the major functions of cyclin D-Cdk4,6 complexes. Cyclin D-Cdk4,6 docks, phosphorylates, and inactivates Rb to promote S Phase. Cyclin D-Cdk4,6 also promotes cell proliferation and survival through phosphorylation of other substrate proteins.[2]

Characterization

This part is validated through 4 experiments: PCR, enzyme digestion, sequencing and functional test.

PCR

The PCR is performed with 2 x Phanta Max Master Mix.

F-Prime: 5’-CTAGCGTTTAAACTTAAGCTTGCCACCATGTCTAAATTCCAGCAGAAGCTTGCG-3’

R-Prime: 5’-GCTTCCGGTCCCGGAGCCTGTTCCGCTCCCTTTTTGTTTTTGCATTCTTGTTCG-3’

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu.

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 cutting procedure was performed with Hind III EcoR I restriction endonuclease bought. 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Fuctional test

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

Figure3. Cell Counting Kit-8 assay showing the 450nm absorbance under 0/24/48/72h cell culturing.

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, indicating our basic part Rb CHelix can bind with CyclinD 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.

[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.