Difference between revisions of "Part:BBa K4414021"
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This composite part consists of a TCE promoter(BBa_K4016011) and a coding sequence of tdTomato(BBa_K4414007).This is a reporter gene that is used to make a visual response to stimulation of upstream genes.
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This composite part consists of an C-Terminal EGFP (BBa_K4414005) and a N-Terminal NR3C1 LBD (BBa_K4414000) domain. It is designed to sense glucocorticoids and locate glucocorticoid receptor (GR) in cells.
  
 
==Usage and Biology==
 
==Usage and Biology==
  
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TCE (BBa_K4016011) consists of 7 repeats of a 19 bp tet operator sequence located upstream of a minimal CMV promoter. In the presence of Dox, Tet-On 3G binds specifically to TCE and activates transcription of the downstream gene of interest (GOI). TCE is the improvement of TRE (BBa_K1431301), The improved TCE promoter shares a similar nucleic acid sequence to the original promoter in TRE, with only a few nucleotides different on the flanking regions of tetO. However, these differences were sufficient to enhance the expression level of GOI.
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We constructed a plasmid to link LBD with the fluorescent protein EGFP to verify the function of LBD. The EGFP on the C-Terminal locates glucocorticoid reporter (GR). The NR3C1 LBD domain on the N-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].  
  
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tdTomato(Tandem-dimer tomato) is a stable, robust red fluorescent protein that is nearly threefold brighter than GFP. Because among all spectral types, the brightest fluorescent protein was Tomato. tdTomato is derived from an intermediate, designated dimer2, that is produced during the disintegration of the tetrameric DsRed protein，it is an orange derivative of the original fruit protein. It needs to be excited at a specific wavelength under a fluorescence microscope to be visible
 
  
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Revision as of 01:29, 9 October 2022


LBD-EGFP

This composite part consists of an C-Terminal EGFP (BBa_K4414005) and a N-Terminal NR3C1 LBD (BBa_K4414000) domain. It is designed to sense glucocorticoids and locate glucocorticoid receptor (GR) in cells.

Usage and Biology

We constructed a plasmid to link LBD with the fluorescent protein EGFP to verify the function of LBD. The EGFP on the C-Terminal locates glucocorticoid reporter (GR). The NR3C1 LBD domain on the N-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].


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Table1. Excitation wavelength and emission wavelength of fluorescent protein tdTomato



In this expression system , tdTomato is equivalent to downstream GOI . When Tet-On 3G binds specifically to TCE, it activates transcription and translation of tdTomato. When this Fluorescent protein reaches a certain amount, We can see it under the fluorescence microscope to qualitatively analyze the expression of upstream genes. In our project, we used it as a downstream part of the response to cortisol stimulation to characterize the magnitude of stress. (Studies have shown that cortisol levels are significantly higher during times of stress than physiological levels.) When the pressure is high, people can easily see the light emitted by the red fluorescent protein tdTomato. This part makes the pressure visualized and also makes our project more practical.



Figure 1. This is a model for the major functions of TCE-tdTomato.When Tet-On 3G binds specifically to TCE, it activates transcription and translation of tdTomato.


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 feasibility and specific effect of this part for constructing the gene pathway of our project, we cotransfected a plasmid with the upstream gene and a plasmid with TCE-Tyr into HEK-293T cells. Cells were treated with 10, 50, or 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. An excitation wave of 554nm was used to make the protein visible under a fluorescence microscope. Then we can observe the red fluorescence of cells at 24 h post glucocorticoids treatment.


Figure 1. This is a model for the major functions of TCE-tdTomato.When Tet-On 3G binds specifically to TCE, it activates transcription and translation of tdTomato.


Result

Figure 4: The red fluorescence of HEK-293T cells at 24 h in the case of 0 and 100 glucocorticoid stimulation.

As it turns out, our pathway is feasible, and we visualized pressure changes that are invisible and difficult to self-accurately assess, characterizing whether there is pressure and the magnitude of pressure in terms of red fluorescence production and strength grade. Future iGEM teams can use this part by simply replace the upstream part to transform the abstract into concrete work as we did.

Reference

[1]Syverud BC, Gumucio JP, Rodriguez BL, Wroblewski OM, Florida SE, Mendias CL, Larkin LM. A Transgenic tdTomato Rat for Cell Migration and Tissue Engineering Applications. Tissue Eng Part C Methods. 2018 May;24(5):263-271. doi: 10.1089/ten.TEC.2017.0406. Epub 2018 Apr 10. PMID: 29490563; PMCID: PMC5946766. [2]Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol. 2004 Dec;22(12):1567-72. doi: 10.1038/nbt1037. Epub 2004 Nov 21. PMID: 15558047.