Difference between revisions of "Part:BBa K4414034"
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Figure1. Schematic figure of PixE-PixD interaction under blue light stimulation
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Figure1. Schematic figure of BBa_K4414034 and BBa_K4414041
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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===Functional Parameters===
 
 
<partinfo>BBa_K4414034 parameters</partinfo>
 
<partinfo>BBa_K4414034 parameters</partinfo>
 
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==Experimental Validation==
 
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We constructed tetR-PixE PixD-Vp64 and TCE-SEAP to test the expression of this part.
 
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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.
 
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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===
 
===Method===

Revision as of 05:50, 9 October 2022


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 BBa_K4414034 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]



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.