Difference between revisions of "Part:BBa K4414031"

 
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__NOTOC__
 
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<partinfo>BBa_K4414031 short</partinfo>
 
<partinfo>BBa_K4414031 short</partinfo>
  
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exchange the position of EGFP and LBD to test function and add the NES sequence to weaken the entry of the GR.
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This composite part consists of an N-Terminal EGFP (BBa_K4414005) and a C-Terminal NR3C1 LBD (BBa_K4414000) domain fused with a NES (BBa_K4414003 ). There is a GSG linker between every two genes. It is designed to sense glucocorticoids and locate glucocorticoid receptor (GR) in cells.
  
  
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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==Usage and Biology==
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The EGFP on the N-Terminal locates glucocorticoid reporter (GR). The NR3C1 LBD domain on the C-Terminal is a 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 trans-activating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression[1]. The NES is a nuclear export signal, which may help us reduce the background values. To ensure that domains work properly, GSG linker is designed to between every two genes.
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===Sequecing===
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The plasmid was sequenced correct.
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
 
<partinfo>BBa_K4414031 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4414031 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K4414031 parameters</partinfo>
 
<partinfo>BBa_K4414031 parameters</partinfo>
 
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==Fuctional test==
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===Method===
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To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding BBa_K4414031. Cells were treated with 10, 50, or 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. Finally, we observe the fluorescence intensity of cells.
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===Result===
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Fluorescence images are shown below, which indicates that glucocorticoids can bind to LBD and enter the nucleus. This provides a basic direction of thinking for our experiments.
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===Reference===
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[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.

Revision as of 01:43, 9 October 2022


EGFP-GSG-NES-GSG-LBD

This composite part consists of an N-Terminal EGFP (BBa_K4414005) and a C-Terminal NR3C1 LBD (BBa_K4414000) domain fused with a NES (BBa_K4414003 ). There is a GSG linker between every two genes. It is designed to sense glucocorticoids and locate glucocorticoid receptor (GR) in cells.


Usage and Biology

The EGFP on the N-Terminal locates glucocorticoid reporter (GR). The NR3C1 LBD domain on the C-Terminal is a 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 trans-activating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression[1]. The NES is a nuclear export signal, which may help us reduce the background values. To ensure that domains work properly, GSG linker is designed to between every two genes.

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]



Fuctional test

Method

To test the ability of this part to respond to glucocorticoids, HEK-293T cells were co-transfected with plasmids encoding BBa_K4414031. Cells were treated with 10, 50, or 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. Finally, we observe the fluorescence intensity of cells.

Result

Fluorescence images are shown below, which indicates that glucocorticoids can bind to LBD and enter the nucleus. This provides a basic direction of thinking for our experiments.

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.