Composite

Part:BBa_K4414028

Designed by: Jiale He   Group: iGEM22_NUDT_CHINA   (2022-09-22)
Revision as of 12:03, 9 October 2022 by TheaYang (Talk | contribs)


LBD-GGGSG-tetR-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. VP64 is a transcriptional activator composed of four tandem copies of VP16 connected with glycine-serine (GS) linkers. 

Figure1.Schematic figure of BBa_K4414028 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-1*GSlinker-Tet R-VP64(BBa_K4414028) 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_K4414028 and (BBa_K4414041).


Result

To validate the function of our composite part Coh2-scFv_cyclinE1(BBa_K4016015) can work with Trim21-DocS(Part: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.

Figure3.Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414028.

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.

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