Part:BBa_K5004001
a tyosinase enzyme
The pigment responsible for hair color in organisms is melanin. We hope to darken hair by promoting melanin synthesis. The Tyr gene encodes a tyrosinase enzyme, which can convert tyrosine into dopa, and then further transform it into dopaquinone. Dopaquinone can subsequently be converted into melanin.
Usage and Biology
We overexpressed Tyr in E. coli BL21 using pSB1A3 plasmid, allowing it to synthesize tyrosinase by lac promoter (Figure 1 and Figure 2). This enzyme, in turn, promotes the darkening of the hair.
Figure 1 Design of the try.
Figure 2 Gel electrophoresis of Try encoding gene.
The utilized DNA Marker is DL2000, which was procured from Takara in Japan. The bands, ordered from highest to lowest, correspond to DNA fragment sizes of 2000 base pairs (bp), 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp, respectively.
Characterization
We initially conducted a validation study on the relationship between melanin content and the substance's light absorption capability (using light with a wavelength of 400nm for the experiment). The results are shown in Figure 3A. It is evident that the more melanin content there is, the stronger the substance's light-absorbing capability. This means that by increasing the melanin content in hair, we can enhance its light absorption ability, making it appear "blacker."
Next, we tested the change in melanin production by the engineered bacteria over time, ensuring its capability to produce melanin and gauging how long it would be appropriate to let the bacteria work. We found that the melanin production increased rapidly within the first 12 hours, after which the growth of melanin content slowed down. By 24 hours, the melanin production was only slightly greater than it was at the 12-hour mark and virtually stopped increasing after that (Figure 3B). This observation can serve as a reference for subsequent, cost-effective synthesis decisions
Figure 3 Experimental results related to melanin.
Subsequently, we attempted to research under which environmental conditions the melanin synthesis is maximized. First, we studied the impact of temperature on the GSH synthesis quantity in Figure 3C. We observed that as the temperature decreased from 47°C to 25°C, the melanin yield over the same duration displayed an initial increase followed by a decrease. The maximum production was achieved at 37°C.
We also studied the effect of pH on melanin production in Figure 3D. We found that as the conditions shifted from neutral to acidic, the melanin yield gradually decreased. This suggests that the most optimal pH for melanin synthesis is around neutral.
Potential application directions
Lactase enzyme has a wide range of application potentials and can be used in lactose intolerance management, dairy product production, and drug synthesis in the future.
References
Pavan, María Elisa, Nancy I. López, and M. Julia Pettinari. "Melanin biosynthesis in bacteria, regulation and production perspectives." Applied Microbiology and Biotechnology 104 (2020): 1357-1370.
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]
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