Difference between revisions of "Part:BBa K4414025"
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This part is an integrated tool for the perception of cortisol stimulation and activates the transcription of the reporter gene.
 
This part is an integrated tool for the perception of cortisol stimulation and activates the transcription of the reporter gene.
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===Sequence and Features===
 
===Sequence and Features===
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===Functional Parameters===
 
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Revision as of 14:24, 8 October 2022


LBD-GGGGGSG-tetR-GGGSG--NLS-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. The NR3C1 LBD domain on the N terminus 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 expression.[1] GGGSG linker, owning some flexibility and allowing the proteins on both sides to complete their own independent functions. Tet R 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. NLS (nuclear localization signal) helps the nucleophilic proteins better move into the nucleus. VP64 is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. 



Figure 1. Schematic figure of BBa_K4414025 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 LBD-GGGGGSG-Tet R-GGGSG-NLS-VP64(BBa_K4414025) and TCE-SEAP(BBa_K4414041). 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 24 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol.[2]

Method

Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol.[2]

Figure2.Schematic representation of the experimental process of validation for BBa_K4414025 and BBa_K4414041.


Result

Results showed similar SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.49 folds).

Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414025.

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.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.