Part:BBa_K4016013

VK_CyclinE1-Trim21-DocS

This composite part is designed to generate cyclinE1 degradation with Part:BBa_K4016012 through DocS-Coh2 interaction and VH_cyclinE1-VK_cyclinE1 folded conformation.

Usage and Biology

In the project this year, we used a previously described ScFv of Cyclin E as our targeting module. With our protein degradation tool, we can degrade cyclin E in a signal-dependent manner, therefore controlling the proliferation of the cells or even synchronize a set of cells at a specific phase of cell cycle.

The design based on the high-affinity protein–protein interaction between two complementary modules: the cohesin and the dockerin. The cohesin–dockerin couple represents the interaction between two complementary families of protein modules that exhibit divergent specificities and affinities..[1]

Upregulation of cyclin E expression begins late in G1 and is maintained into S-phase.[2] It is a critical cell cycle protein in the regulated progression of normal cells to replicate their DNA. Ectopic overexpression of cyclin E results in accelerated G1 progression, chromosome instability, and a reduced requirement for growthfactors..[3]

Because expressing the full-length of ScFv might already be sufficient to trigger the dysfunction of cyclins, we designed another set of constructs that split the ScFv into VH and VK fragments, and fused these fragments on either N terminus of C terminus of Predator proteins. In this case, the small molecular induced dimerization would also trigger the reconstitution of ScFv, therefore reducing the potential effect of full-length ScFv on Cyclins.

In the design of BBa_K4016013 we link Docs and Trim21 with VH fragment by the short flexible polypeptide linker to prove if the VK and VH fragments works toghther well.

Figure 1. Schematic figure of BBa_K4016013 and BBa_K4016012.

Characterization

This part BBa_K4016013 was cloned in pXQ136 plasmid and transfected into HEK293T cell lines using Invitrogen LipofectamineTM 3000.This part is validated through four ways: PCR, Sequence, and functional testing.

PCR

The PCR is performed with Green Taq Mix by Vazyme.

F-Prime: 5’-CTAGCGTTTAAACTTAAGCTTGCCACCATGGATATAGTGCTGACCCAAAG-3’

R-Prime: 5’- TGGATATCTGCAGAATTCTTAACCtggagaggtaatatccaggtcct-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 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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 37
  Illegal NheI site found at 525
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1194
  Illegal BamHI site found at 1732
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 482
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 60
  Illegal BsaI site found at 1572
  

Fuctional test

This part (BBa_K4016013) was tested together with BBa_K4016012

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 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. Schematic representation of the experimental process of validation for BBa_K4016012 and [[Part:BBa_K4016013]] ===Result===

Figure 3.CK8 cell proliferation assay of HEK-293 cells transfected with indicated plasmids. Results in figure3 shows the reduction of cell proliferation was unsatisfying. The 450nm absorbance of experimental group transfected with pcDNA3.1-VH_CyclinE1-Coh2 and pcDNA3.1-VK_CyclinE1-Trim21-DocS is slightly higher than the control gruop,indicating that the design of split expression of VH and VK of cyclinE_scFv didn't work as we expected. ===Reference=== [1]Jiang, Hongyu. Expression and purification of a human anti-cyclinD1 single-chain variable fragment antibody AD5 and its characterization[J]. International Journal of Molecular Medicine, 2013, 32(6):1451-1457. [2]Stacey M. Ivanchuk, James T. Rutka,CHAPTER 9 - Regulation of the Cell Cycle and Interventional Developmental Therapeutics. HERBERT B. NEWTON, Handbook of Brain Tumor Chemotherapy, Academic Press, 2006, 123-140. [3]Randall W. Strube , Si-Yi Chen, et al. Characterization of anti-cyclin E single-chain Fv antibodies and intrabodies in breast cancer cells: enhanced intracellular stability of novel sFv–Fc intrabodies[J].Journal of Immunological Methods, 2002, 263: 149–167.