Difference between revisions of "Part:BBa K4016015"

Revision as of 19:11, 20 October 2021

Coh2-ScFv_CyclinE1

This composite part is designed to generate cyclinE1 degradation with Part:BBa_K3396005 through DocS-Coh2 interaction and ScFv_CyclinE1-CyclinE1 connection. With this composite part, ScFv_CyclinE1 and Coh2 can be expressed in cells, and ScFv_CyclinE1 was added to N-terminal of Coh2. Compared with the ScFv_CyclinE1-Coh2(Part:BBa_K4016014), this composite part changed the positions of Coh2 and Scfv_CyclinE1.

Usage and Biology

To realize the goal of degradation, we designed the composite part Coh2-ScFv_CyclinE1, and used the function and benefits of ScFv to target cyclinE1.

Toward the goal of developing a system to block cyclin E function in normal and breast cancer cells, Randall W. Strube and his team have developed anti-cyclin E single-chain antibodies (sFvs) for use as intrabodies. Randall W. Strube and his team have cloned the variable region genes from two hybridoma cell lines that produce anti-human cyclin E antibodies, linked them into sFvs, and showed their ability to bind cyclin E when expressed as sFv – Fcfusion proteins.[1]

The Coh2 module binds DocS (Part:BBa_K3396000) modules constitutively. The Coh2[2] module comes from The C. thermocellum scaffoldin 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.

Our team used the mechanism of specific binding between Scfv which included parts of the variable regions of heavy (VH) and light chains (VL) fused together to form a single polypeptide and its target protein cyclinE1. Based on the feasibility of Coh2-Docs, our system can degrade cyclinE1 to control the cell cycle.

Special design

Comparing the two parts ScFv_CyclinE1-Coh2 and Coh2-ScFv_CyclinE1, we can find that it just changed the position of two basic parts. The purpose of this design is to explore whether the changes in the front and back positions of the two different basic parts in the plasmid will affect the function or even the activity of the protein.

Figure 1. Schematic figure of BBa_K4016015

Here is the mechanism :

1.Trim21-DocS connect with Coh2-Scfv_CyclinE1 or Scfv_CyclinE1-Coh2 through DocS-Coh2 interaction and forms a dimerized complex

2.Inside the complex, Scfv_CyclinE1 and then targets cyclinE1

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

Characterization

This part was validated through four ways:PCR, enzyme digestion, sequencing and functional test.

PCR

The PCR is performed with Green Taq Mix by Vazyme.

F-Prime:5’-CTAGCGTTTAAACTTAAGCTTGCCACCATGGTGGTGGTGGAGATCGGCAAG-3’

R-Prime: 5’-TGGATATCTGCAGAATTCTTATCTTTTGATCTCAAGCTTGGT-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 gel.

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 856
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 799
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 879

Functional test

This part was tested together with ScFv_CyclinE1-Coh2 (Part:BBa_K4016014)

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.

Result

To validate the function of our composite part Coh2-scFv_cyclinE1(BBa_K4016015) can work with Trim21-DocS(BBa_K3396005), we did a CCK-8 assay, more accurately a cell proliferation assay. The results showed that after 24 hours, 48 hours, and 72 hours, the cell proliferation trend of our system was weaker than that of the control group, indicating that our system can indeed degrade CyclinE1, thereby regulate the cell cycle, ultimately inhibit cell proliferation to a certain extent.

Figure 2. CCK-8 assay showing the result of cell proliferation under the two different conditions in different stages. The results of cell proliferation was presented by absorbance. This experiment was run in two conditions, with our system and and empty vector.

Compared with the experimental results of scFv_cyclinE1-Coh2, the results of Coh2-scFv_cyclinE1 had more obvious changes than the control group, which showed that the design of Coh2-scFv_cyclinE1 was more effective for our system.

Reference

[1]Randall W. Strube,Si-Yi Chen. Characterization of anti-cyclin E single-chain Fv antibodies and intrabodies in breast cancer cells: enhanced intracellular stability of novel sFv–F c intrabodies[J]. Journal of Immunological Methods,2002,263(1):

[2] BARAK Y, HANDELSMAN T, NAKAR D, et al. Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction [J]. J Mol Recognit, 2005, 18(6): 491-501.

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-Figure 2. CCK-8 assay showing the result of cell proliferation under the three different conditions in different stages. The results of cell proliferation was presented by absorbance. This experiment was run in three conditions, and the data represent results obtained from three independent experiments. The experiment not only showed the work effect of our system compared with the control experiment in different periods, but also compared the effects of two different designs.
+Figure 2. CCK-8 assay showing the result of cell proliferation under the two different conditions in different stages. The results of cell proliferation was presented by absorbance. This experiment was run in two conditions, with our system and and empty vector.
 Compared with the experimental results of scFv_cyclinE1-Coh2, the results of Coh2-scFv_cyclinE1 had more obvious changes than the control group, which showed that the design of Coh2-scFv_cyclinE1 was more effective for our system.
 ===Reference===