Difference between revisions of "Part:BBa K4414040"
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<partinfo>BBa_K4016012 short</partinfo>
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<partinfo>BBa_K4414040 short</partinfo>
  
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This composite part is designed to generate cyclinE1 degradation with [[Part:BBa_K4016013]] through DocS-Coh2 interaction and VH_cyclinE1-VK_cyclinE1 folded conformation.
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This part is an integrated tool for the perception of cortisol stimulation and activates the transcription of the reporter gene.
  
  
 
==Usage and Biology==
 
==Usage and Biology==
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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.
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As a glucocorticoid sensor, this part is designed to enter the nucleus upon glucocorticoid stimulation and bind to the TCE promoter to activate downstream transcription.
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Tet R on the N terminus in our design provides DNA binding domain tightly binding to the downstream gene, which binds to the TCE promoter (BBa_K4016011) consisting of seven direct 19-bp Tet operator sequence (Teto) repeats. The NR3C1 LBD domain is the ligand binding domain of the glucocorticoid receptor (GR). This LBD domain can translocate the fusion protein into the nucleus upon glucocorticoid stimulation. It also has a transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene 1) GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. VP64 on the C terminus is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. 
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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]
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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]
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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.  
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In the design of BBa_K4016012 we link Coh2 with VH fragment by a short flexible polypeptide linker, so that it can work as a whole.
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Figure 1. Schematic Figure of BBa_K4016012 and BBa_K4016013
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Figure1.Schematic figure of BBa_K4414040 and BBa_K4414041.
  
  
  
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==Characterization==
 
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This part was cloned in pXQ135 plasmid and transfected into HEK293T cell lines using Invitrogen LipofectamineTM 3000.It was validated through four ways:PCR, enzyme digestion, sequencing and functional test.
 
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===PCR===
 
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The PCR is performed with Green Taq Mix by Vazyme.
 
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F-Prime: 5’-CTAGCGTTTAAACTTAAGCTTGCCACCATGGAGGTTCAACTGGAGGAGTCTG-3’
 
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R-Prime: 5’- TGGATATCTGCAGAATTCTTACACGTTCACGCCGCCGTCGAT-3’
 
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The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose gel.
 
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===Enzyme Digestion===
 
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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.
 
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The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.
 
  
  
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===Sequence and Features===
 
===Sequence and Features===
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<partinfo>BBa_K4016012 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K4414040 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K4016012 parameters</partinfo>
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<partinfo>BBa_K4414040 parameters</partinfo>
 
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==Functional test==
 
==Functional test==
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This part (BBa_K4016012) was tested together with BBa_K4016013
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To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both Tet R-LBD-1xGS linker-VP64(BBa_K4414040) and TCE-SEAP(BBa_K4414041).
  
 
===Method===
 
===Method===
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*1. Cell transfection
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Cells were treated with 0 or 100 nm Glucocorticoids 6h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24h post glucocorticoids treatment. SEAP activity was measured according to a published protocol. [2]
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(1)Seed HEK293T cells into 6-well cell culture plates.
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(2)Culture for 16 h before transfection  
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(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)
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(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.
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(5)Cells are then changed into fresh medium and culture for 18 h before subculture.
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*2.CCK-8 assay
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(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.
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(2)Dispense 100ul of cell suspension (approximately 30000 cells per well) into 96 well plates.
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(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
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(4)Add 10 ul CCK-8 solution to each well and incubate for 2 h in the incubator.
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(5)Record results using microplate reader to measure the absorbance at 450 nm.
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Figure2. Schematic representation of the experimental process of validation for BBa_K4016012 and [[Part:BBa_K4016013]]
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Figure2.Schematic representation of the experimental process of validation for BBa_K4414040 and BBa_K4414041.
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Figure 3.CK8 cell proliferation assay of HEK-293 cells transfected with indicated plasmids.
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Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414040.  
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===Reference===
 
===Reference===
  
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[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.
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[1].Weikum ER, Knuesel MT, Ortlund EA, Yamamoto KR. Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat Rev Mol Cell Biol. 2017 Mar;18(3):159-174. doi: 10.1038/nrm.2016.152. Epub 2017 Jan 5. PMID: 28053348; PMCID: PMC6257982.
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[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.
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[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.
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[2].Shao J, Qiu X, Xie M. Engineering Mammalian Cells to Control Glucose Homeostasis. Methods Mol Biol. 2021;2312:35-57. doi: 10.1007/978-1-0716-1441-9_3. PMID: 34228283.

Revision as of 14:44, 8 October 2022


TetR-GGGSG-LBD-GGGSG-VP64

This part is an integrated tool for the perception of cortisol stimulation and activates the transcription of the reporter gene.


Usage and Biology

As a glucocorticoid sensor, this part is designed to enter the nucleus upon glucocorticoid stimulation and bind to the TCE promoter to activate downstream transcription. Tet R on the N terminus in our design provides DNA binding domain tightly binding to the downstream gene, which binds to the TCE promoter (BBa_K4016011) consisting of seven direct 19-bp Tet operator sequence (Teto) repeats. The NR3C1 LBD domain is the ligand binding domain of the glucocorticoid receptor (GR). This LBD domain can translocate the fusion protein into the nucleus upon glucocorticoid stimulation. It also has a transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene 1) GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. VP64 on the C terminus is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. 

Figure1.Schematic figure of BBa_K4414040 and BBa_K4414041.



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]



Functional test

To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding both Tet R-LBD-1xGS linker-VP64(BBa_K4414040) and TCE-SEAP(BBa_K4414041).

Method

Cells were treated with 0 or 100 nm Glucocorticoids 6h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24h post glucocorticoids treatment. SEAP activity was measured according to a published protocol. [2]

Figure2.Schematic representation of the experimental process of validation for BBa_K4414040 and BBa_K4414041.


Result

Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414040.

Reference

[1].Weikum ER, Knuesel MT, Ortlund EA, Yamamoto KR. Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat Rev Mol Cell Biol. 2017 Mar;18(3):159-174. doi: 10.1038/nrm.2016.152. Epub 2017 Jan 5. PMID: 28053348; PMCID: PMC6257982.

[2].Shao J, Qiu X, Xie M. Engineering Mammalian Cells to Control Glucose Homeostasis. Methods Mol Biol. 2021;2312:35-57. doi: 10.1007/978-1-0716-1441-9_3. PMID: 34228283.