Part:BBa_K4016037

ScFv_CyclinE1-CIB1

This composite part is designed to generate cyclinE1 degradation with Trim21-CRY2(Part:BBa_K4016035) through CRY2-CIB1 dimerization.

Usage and Biology

This part is composed of ScFv_CyclinE1 by CIB1. When induced by blue light, CIB1 dimerizes with its binding partner CRY2, effectively bringing the target site defined Trim21. While achieving the purpose of optical control, the ScFv-cyclinE1 bind with both Trim21 and cyclinE1 to prove our results.

Figure 1. Schematic figure of BBa_K4016037

• Here is the mechanism of the recombined ScFv_CyclinE1-CIB1:

1.ScFv_CyclinE1-CIB1 connect with Trim21-CRY2 through CRY2-CIB1 interaction and forms a dimerized complex.

2.Inside the complex, ScFv_CyclinE1 targets cyclinE1.

3.Cyclin E1 is degraded by ubiquitin-proteasome system recruited by Trim21

Characterization

This part is validated through four ways: colony PCR, sequencing and functional testing.

Colony PCR

The PCR is performed with Green Taq Mix by Vazyme.

F-Prime: 5’-CTAGCGTTTAAACTTAAGCTTGCCACCATGGAGGTTCAACTGGAGGAGTCTG-3’

R-Prime: 5’-TGGATATCTGCAGAATTCTTAttaGATGTAGTCGGTCTTCTC-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
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 838
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional test

This part was tested together with or Trim21-CRY2(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.

Reference

1.Kennedy, M. J. et al. Rapid blue-light–mediated induction of protein interactions in living cells. Nat Methods 7, 973–975 (2010).

2.Bugaj, L. J., Choksi, A. T., Mesuda, C. K., Kane, R. S. & Schaffer, D. V. Optogenetic protein clustering and signaling activation in mammalian cells. Nat Methods 10, 249–252 (2013).

3.Taslimi, A. et al. Optimized second-generation CRY2–CIB dimerizers and photoactivatable Cre recombinase. Nat Chem Biol 12, 425–430 (2016).