Part:BBa_K4414017

TCE-TYR

This composite part consists of a TCE promoter(Part:BBa_K4016011) and a coding sequence of TYR(Part:BBa_K4414006).This is a reporter gene that is used to make a visual response to stimulation of upstream genes.

Usage and Biology

TCE (Part: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 (Part: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.

TYR (Part:BBa_K4414006) is a gene encoding Tyrosinase .Tyrosinase is a key enzyme in melanin synthesis that catalyzes the rate-limiting step of the reaction. By activating tyrosine kinase can increase the yield of melanin, forming melanin deposition(Hearing & Jiménez, 1987) .

In this expression system , TYR is equivalent to downstream GOI . When Tet-On 3G binds specifically to TCE, transcription of TYR is activated to generate melanin. When melanin reaches a certain amount, we can directly see it with the naked eye to qualitatively analyze the expression of upstream genes, without the need for preliminary detection by professional instruments, which greatly improves the simplicity of detection.(Figure 1) 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 melanin catalyzed by TYR encoded enzymes. This part makes the pressure visualized and also makes our project more practical.

Figure1. This is a model for the major functions of TCE-TYR.When Tet-On 3G binds specifically to TCE, it activates transcription and translation of TYR.

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]

Functional Validation

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.

Method

Cells were treated with 1, 10, 50, or 100 nM Glucocorticoids 6 h post-transfection. Cells without glucocorticoid treatment were used as control. Observe the blackening of cells at 24 h or 48 h post glucocorticoids treatment.

Figure 2: Cotransfected our upstream plasmid with the upstream gene and a plasmid with TCE-TYR into cells

Result

The test results are as follows:

Figure 3: (Left)The blackening of HEK-293T cells at 24 h or 48 h post glucocorticoids treatment in microscope.(Right)The blackening of HEK-293T cells at 24 h or 48 h post glucocorticoids treatment under the naked eye.

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 melanin 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. Hearing, V. J., & Jiménez, M. (1987). Mammalian tyrosinase—The critical regulatory control point in melanocyte pigmentation. The International Journal of Biochemistry, 19(12), 1141–1147. https://doi.org/10.1016/0020-711x(87)90095-4