Part:BBa_K4016018

VK_CyclinE1-Docs-Trim21

This composite part consists of truncated VK_cyclinE1 (Part:BBa_K4016007) fused in the N-terminal, Trim21 (Part:BBa_K3396007) fused in the C-terminal and DocS in the middle. It is designed to generate cyclinE1 degradation with Part:BBa_K4016016 through DocS-Coh2 interaction and VH_cyclinE1-VK_cyclinE1 folded conformation.

Usage and Biology

The DocS module comes from C. thermocellum and it could recognize and bind tightly to complementary Coh2 modules. 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.[1]

Trim 21 comes from the family of TRIM. In iGEM2020, we truncated the Trim21 protein to maintain its RING domain, B box domain and coiled coli domain, while the antibody binding PRYSPRY domain was removed. This truncated protein maintained the E3 ubiquitin ligase activity, and provided an open interface for other protein dimerization pairs to be added. (link to BBa_K3396007)

Cyclin E 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 growth factors. [2] Therefore, our team center on degrading cyclinE1 to regulate cell cycle.

VK_cyclinE1 comes from hybridoma-rearranged heavy chain variable region gene in HE12 and HE172 hybridomas. We use this part together with Coh2-VH_cyclinE1 to specifically recognize and degrade cyclinE1.

Figure 1. Schematic figure of BBa_K4016018 and BBa_K4016016.

• Here is the mechanism of the recombined VK_cyclinE1-DocS-Trim21

1.Trim21-DocS-VK_cyclinE1 connect with Coh2-VH_cyclinE1 through DocS-Coh2 interaction and forms a dimerized complex

2.Inside the complex, VH_cyclinE1 combines with VK_cyclinE1 and then targets cyclinE1

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

Special design

Click here (Part:BBa_K4016016) to see the detailed reason why we separately express VH and VK of cyclinE1. What is more, we tried to find out the optimized spatial structure of this complex by switching the position of truncated Trim21 and VK_cyclinE1 compared with Part:BBa_K4016017. Therefore, we can choose the best structure according to their functional test result and introduce the blue light-induced interaction pairs such as CRY2-CIB1 into the system by replacing DocS-Coh2 interaction.

Characterization

This part is validated through four ways: colony PCR, enzyme digestion and sequencing..

PCR

The PCR is performed with Green Taq Mix by Vazyme.

F-Prime: 5’- CTAGCGTTTAAACTTAAGCTTGCCACCATGgcttcagcagcacgcttg-3’

R-Prime: 5’- TGGATATCTGCAGAATTCTTATCTTTTGATCTCAAGCTTGG-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 735
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1404
  Illegal BamHI site found at 1942
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 692
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 60
  Illegal BsaI site found at 1782
  

Reference

[1]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:10.1002/jmr.749 (2005)

[2] 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.