Part:BBa_K4016016

Coh2-VH_CyclinE1

This composite part is designed to generate cyclinE1 degradation with Part:BBa_K4016017 or Part:BBa_K4016018 through DocS-Coh2 interaction and VH_cyclinE1-VK_cyclinE1 folded conformation.

Usage and Biology

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. 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]

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.

VH_cyclinE1 comes from hybridoma-rearranged heavy chain variable region gene in HE12 and HE172 hybridomas. By the same way, VK_cyclinE1 is produced from light chain variable region gene. We use this part together with Trim21-DocS-VK_cyclinE1(Part:BBa_K4016017) or VK_cyclinE1-DocS-Trim21(Part:BBa_K4016018) to specifically recognize and degrade cyclinE1.

Figure 1. Schematic figure of BBa_K4016016

• Here is the mechanism of the recombined Coh2-VH_cyclinE1:

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

In case that full length scFv of cyclinE1 would directly interact with cyclinE1 and lead to negative impact on cell proliferation, we separately clone the heavy chain variable region (VH) gene and light chain variable region (VK) gene and assume that VH and VK will take on a folded conformation when they are adjacent in three-dimensional space, and thus recognize and bind to CyclinE1 tightly and specifically. Under this circumstance, the targeting module would also be assembled to make a complete Predator system avoiding the high-background problem of expressing full length scFv of cyclinE1 once introduced into cells. Meanwhile, we utilize the well-characterized and already-tested parts Coh2 (Part:BBa_K3396001) and DocS (Part:BBa_K3396000) to verify the strategy of separate translation of VH and VK. The positive result lays the foundation for replacing the DocS-Coh2 interaction with blue light-inducible CRY2-CIB1 interaction.

Characterization

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

Colony PCR

The PCR is performed with Green Taq Mix by Vazyme.

F-Prime: 5’-CTAGCGTTTAAACTTAAGCTTGCCACCATGGTGGTGGTGGAGATCGGCAAG-3’

R-Prime: 5’-TGGATATCTGCAGAATTCTTACGCGGACACTGTCACTAATG-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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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.