Composite

Part:BBa_K4414037

Designed by: Xiyao Yang   Group: iGEM22_NUDT_CHINA   (2022-09-22)
Revision as of 03:41, 9 October 2022 by TheaYang (Talk | contribs)


TetR-GSG-NES-GSG-LBD

This composite part consists of a C-terminal tetR(BBa_K4414009) domain and an NR3C1 LBD(BBa_K4414000) domain fused with NES(BBa_K4414003). It is designed to sense glucocorticoids 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. This part consists of a tetR DNA binding domain, which binds to the TCE promoter (BBa_K4016011) consisting of seven direct 19-bp tet operator sequence (tetO) repeats. The NR3C1 LBD domain on the N terminal 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] NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol .



Figure1. Figure1. Schematic figure of BBa_K4414037 and BBa_K4414041


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 Validation

Method

HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414037 and TCE-SEAP. Cells were treated with 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. Culture medium was collected at 24 h or 48 h post glucocorticoids treatment. SEAP activity was measured according to a published protocol. [2]

Figure 2


Result

The test results are as follows:

Figure3. Glucocorticoid-stimulated transcriptional activation of SEAP mediated by BBa_K4414037.

Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control. A dose dependence was observed within 0-100 nM of glucocorticoid (Figure 3).

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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