Part:BBa_K4414021
TCE-tdTomato
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.
Usage and Biology
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.
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
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.
Sequecing
The plasmid was sequenced correct.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
Result
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.
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