Composite

Part:BBa_K4414034

Designed by: Shiyin Jin   Group: iGEM22_NUDT_CHINA   (2022-09-22)
Revision as of 15:48, 8 October 2022 by Jinshiyin (Talk | contribs)


TetR-LBD

This composite part consists of an N-terminal tetR(BBa_K4414009) domain and a C-terminal NR3C1 LBD(BBa_K4414000) domain without GS linker. 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 C 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]

Figure1. Schematic figure of PixE-PixD interaction under blue light stimulation




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]



Experimental Validation

We constructed tetR-PixE PixD-Vp64 and TCE-SEAP to test the expression of this part. VP64 is a transcriptional activator. When fused to another protein domain that can bind near the promoter of a gene, VP64 acts as a strong transcriptional activator. TetR can recognize and combine with TCE then inhibit its downstream transcription. The interaction of PixD and PixE would restrain the TCE’s inhibition and start the transcription of SEAP.

Method

HEK-293T cells were co-transfected with plasmids encoding both BBa_K4414034 and TCE-SEAP(BBa_K4414041). 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 post glucocorticoids treatment. SEAP activity was measured according to a published protocol. [2]


Result


Results showed significantly increased SEAP expression in glucocorticoid-treated cells compared to the non-treated control (1.29 folds)(Figure 2).



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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