Part:BBa_K3633000

Coding sequence for tyrosinase from B. megaterium

Description

Fig 1. Melanin Synthesis Pathway

Tyr gene codes for the enzyme tyrosinase, which is located in melanocytes in our skin. Melanocytes are responsible for producing melanin. Melanin is responsible for giving our skin and hair color. It includes eumelanin and pheomelanin. Eumelanin usually shows darker color than pheomelanin. As shown below, tyrosinase is responsible for the first step in melanin production.

Tyrosine is one of the 20 standard amino acids present in the body and is used by cells to synthesize proteins. This is a non-essential amino acid, meaning that it is also synthesized by the body when it is not only ingested in one's diet. Tyrosine is found in casein, a protein in milk and other milk-based products (like cheese). Tyrosine is also present in non-dairy foods that are aged, including some types of meats and red wine.

Experiments & Results

Successful production in E.coli BL21(DE3) and vibrio natriegens with promoter J23102

We constructed the plasmid J23102-Tyr1 and successfully transformed the plasmid into E. coli BL21(DE3) and Vibrio natriegens ATCC 14048. We added 2.5 mg L-DOPA and 0.4 mg L-Tyrosine as substrates and cultured the bacteria in shakers (220 rpm) at 25℃ or 37℃.

Fig 2. (A) Production of melanin in E. coli BL21(DE3) and Vibrio natriegens at 25℃ and 37℃ in 72 h. (B) Production of melanin from 0-72 h. Top left: E. coli BL21(DE3), 25℃. Top right: E. coli BL21(DE3), 37℃. Bottom left: Vibrio natriegens, 25℃. Bottom right: Vibrio natriegens, 37℃.

Both bacteria at 37℃ produced more rapidly than bacteria at 25℃. V. natriegens produced slightly faster at first (0-36 h), but was then exceed by E. coli. Both types of bacteria in both conditions reached the maximum production at 48-60 h, which may be due to lack of substrates or nutrition. E. coli could produce more melanin than V. natriegens by the end of 72 h. The highest production, 11.8 g/L, was achieved by E. coli BL21(DE3) at 37℃.

Successful production in E.coli BL21(DE3) and vibrio natriegens with promoter pTac

We constructed the plasmid pTac-Tyr1 and successfully transformed the plasmid into E. coli BL21(DE3) and Vibrio natriegens ATCC 14048. We added 2.5 mg L-DOPA, 0.4 mg L-Tyrosine, and 1mM IPTG as substrates and cultured the bacteria in shakers (220 rpm) at 37℃. The result shows that melanin was produced. However, the effect of the pTac promoter is not as good as the constitutive promoter J23102.

Fig 3.Production of melanin in E. coli BL21(DE3) and Vibrio natriegens at 37℃ in 78 h. The horizontal axis is time (hours), and the vertical axis is the absorbance of the bacterial solution at 400 nm.

Hair dye using melanin produced by engineering E.coli(J23102 promoter)

We first incubated the hair in pH=9 Ca(OH)2 at 50℃ for 40 min. Next, we added the melanin bacterial solution and kept at 50℃ for another 40 min. We successfully dyed hair with our synthesized pigment.

Fig 4. Hair dye results with synthesized pigments including melanin hair dye, which is successful, with positive and blank controls.

Sequence & Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 320
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

References

1. 赵飞龙, 徐亚军, "微生物黑色素的研究", 中国调味品, vol. 34, no. 9, 2009.

2. Z. Wang et al., "Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application", Applied and Environmental Microbiology, vol. 86, no. 5, 2019. Available: 10.1128/aem.02749-19.

3. M. Pavan, N. López and M. Pettinari, "Melanin biosynthesis in bacteria, regulation and production perspectives", Applied Microbiology and Biotechnology, vol. 104, no. 4, pp. 1357-1370, 2019. Available: 10.1007/s00253-019-10245-y.

4. "Team:NYMU-Taipei/Pigments - 2017.igem.org", 2017.igem.org, 2020. [Online]Available: http://2017.igem.org/Team:NYMU-Taipei/Pigments. [Accessed: Jun-2020].