Part:BBa_K5241010

GA11-mel-CYC1

Short Description:In Pseudomonas maltophilia, the mel gene encodes tyrosinase, which is one of the key enzymes in melanin synthesis. This enzyme catalyzes the conversion of tyrosine to L-DOPA and ultimately forms melanin through a series of oxidation reactions.

Gene Circuit Diagram:

Figure 1 The genetic circuit diagram of thebrewing yeast transformed with the mel gene.

Source:Pseudomonas maltophilia

Description:

The yeast galactokinase (GAL1) gene is a strongly inducible promoter and is the most commonly used promoter in the yeast recombinant protein expression system. The transcriptional activity of the GAL1 promoter is related to the carbon source in the culture medium. In the presence of glucose, the transcription of the GAL1 promoter is inhibited; galactose, on the other hand, activates the promoter. Typically, after about 4 hours of induction with galactose, the expression of recombinant proteins can be detected in cells cultured with glucose. The role of the mel gene in brewing yeast is mainly as a reporter gene. The tyrosinase enzyme it encodes can catalyze the conversion of tyrosine to dopamine, which is then further converted into melanin. This produced melanin is non-toxic and harmless, allowing for the direct observation of a black phenotype on the culture medium without the need to add color-developing compounds or use special instruments.

The melanin expressed by the mel gene in brewing yeast appears red, possibly because a larger amount of pheomelanin is produced.

Result

Figure 2 The transformation of brewing yeast with mel gene.

The transformation of brewing yeast with mel gene

<1>Measuring the radiation resistance of brewing yeast expressing melanin using UV irradiation experiment.

Preliminary experiments on ultraviolet lethality were conducted using red yeast expressing the mel gene.

. After spreading on YPD agar plates, the yeast was exposed to 254+302+365nm UV radiation with the lid on, and then incubated in a 30-degree incubator until colonies were visible (total of 24 hours), and then observed and photographed.

Figure 3 The survival of yeast strains on plates after UV irradiation.

Figure 4 The trend graph of lethality of red yeast after UV irradiation.

The Saccharomyces cerevisiae that we have engineered possesses certain radiation-resistant effects.

<2>The optimization of UV irradiation experiments.

We realized that using a single colony for UV irradiation experiments was inadequate for presenting complete results. Therefore, we added a control group with normal yeast that had not been transformed with melanin and conducted UV irradiation experiments.The survival of yeast expressing the mel gene, which produces melanin, and normal yeast after exposure to ultraviolet light with an intensity of 168.5 μW/cm² is as follows.

(Ultraviolet irradiation of 168.5μW/cm²)Ultraviolet irradiation time.	Saccharomyces cerevisiae （transformed with the mel gene survival）	Saccharomyces cerevisiae transformed with the mel gene survival(Experimental results figure)	Saccharomyces cerevisiae survival(Not transformed.)	Saccharomyces cerevisiae survival(Experimental results figure)
0h	160		88
2h	117		62
4h	115		18
8h	109		4

Figure 5 The survival of yeast expressing the mel gene, which produces melanin, and normal yeast after exposure to ultraviolet light with an intensity of 168.5 μW/cm².

Survival rate
time/h	Saccharomyces cerevisiae expressing the mel gene.	Non-transformed brewer’s yeast.
0	1	1
2	0.73125	0.704545455
4	0.71875	0.204545455
8	0.68125	0.045454545

Table 1 The survival rates of Saccharomyces cerevisiae transformed with mel and non-transformed Saccharomyces cerevisiae under UV irradiation, as calculated from figure 6.

Figure 6 Survival of bacterial strains under UV irradiation.

From Figure , it is known that the survival rate of strains expressing melanin after exposure to ultraviolet light with an intensity of 168.5 μW/cm² is higher than that of normal strains.

Reference documentation

[1] Ruohola, H., Liljeström, P. L., Torkkeli, T., Kopu, H., Lehtinen, P., Kalkkinen, N., & Korhola, M. (1986). Expression and regulation of the yeast MEL1 gene. FEMS Microbiology Letters, 34(2), 179–185.

[2] Naumov, G., Naumova, E., Turakainen, H., Suominen, P., & Korhola, M. (1990). A new family of polymorphic genes in Saccharomyces cerevisiae: alpha-galactosidase genes MEL1-MEL7. Molecular Genetics and Genomics, 224(1), 119–128.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 150
  
• 1000
  COMPATIBLE WITH RFC[1000]