Team:Shanghai SFLS SPBS/Results
Overview
We tried to synthesize pigments melanin, indigo, dopaxanthin, and indoline-betacyanin by engineering the metabolic pathways of bacteria E. coli and Vibrio natriegens. We successfully produced melanin in both bacteria, indigo in E. coli DH5α, and dopaxanthin and indoline-betacyanin in IPTG-induced E. coli BL21(DE3).
Additionally, inspired by the conventional hair dye method that uses pigment precursors combined with oxidants, we thought of combining pigment precursors with an oxidase, laccase. We produced dopamine - most of which, unfortunately, polymerized into polydopamine - and bacterial laccase. We successfully extracted laccase, performed SDS-PAGE electrophoresis, and conducted an enzyme activity assay.
To analyze the production level of the pigments and pigment precursors, we used a microplate reader. We first used standard pigments or precursors to measure the absorbance spectrum and draw the standard curve. Then, after we produced the pigments, we measured the absorbance of acquired pigments and estimated their concentrations. We were later able to borrow HPLC-MS equipment from Shimadzu Enterprise Management (China) Co., Ltd. and used HPLC-MS to measure the production levels more accurately.
Finally, we tried to dye hair with standard (directly bought on the Internet) and synthesized pigments. We successfully dyed hair with all standard pigments, as well as synthesized melanin and indoline-betacyanin. It is worth noting that the dyeing effect of synthesized indoline-betacyanin is better than that of standard betacyanins.
On this page, we will first discuss our pre-experiment to measure the standard curve of pigments and dopamine (after polymerization to polydopamine). Next, we will discuss the production of pigments, pigment precursor dopamine, and laccase. Finally, our hair dye results will be presented, and future goals will be discussed.
Standard curve of pigments and pigment precursor dopamine
To measure the production of synthesized pigments, we measured the standard curves of pigments and pigment precursor dopamine. We bought melanin, indigo, betacyanins, and dopamine hydrochloride at Sangon Biotech. To acquire the standard curve, we first measured the absorbance spectrum of the pigments and identified the peak absorbance. Then, we acquired solutions of different concentrations and measured their absorbance at the peak absorbance wavelength. Finally, we drew the standard curve. With the standard curves, we will be able to estimate the level production with the absorbance of our synthesized pigments (after dilution or concentration, if necessary).
Melanin
According to the article, melanin has the greatest absorbance at 400nm though it absorbs almost all the visible lights. We confect melanin solution dissolved in DMSO of 1000μg/mL, 500μg/mL, 100μg/mL, 50μg/mL, 10μg/mL, 1μg/mL. It shows a linear relationship between the absorbance and the concentration in this region.
Figure 1. Standard curve of absorbance of melanin. The result shows a linear relationship between the absorbance and concentration in this region.
Indigo
Indigo has the greatest absorbance at 605nm. Since the absorbance doesn't have a linear relationship with the concentration when indigo concentration is too high, we confect indigo solution dissolved in DMSO of 100μg/mL, 50μg/mL, 10μg/mL, 5μg/mL, 1μg/mL.
Figure 2. Standard curve of absorbance of indigo. The result shows a linear relationship between the absorbance and concentration in this region.
Betacyanin
Betacyanin has the greatest absorbance at 530nm. We confect betacyanin solution dissolved in water of 10mg/ml, 5mg/ml, 1mg/ml, 0.5mg/ml, 0.1mg/ml. Since there are different kinds of betacyanins based on the different substrates, the betacyanin we bought may not be identical to the indoline-betacyanin we synthesized, which suggests deviation of the result from the graph. It is also noted that since we didn't find means to buy betaxanthin, the standard curve of betaxanthin is not constructed and we would calculate the relative value of the concentration of betaxanthin.
Figure 3. Standard curve of absorbance of betacyanin. The result shows a linear relationship between the absorbance and concentration in this region. Since there are different kinds of betacyanins based on the different substrates, the betacyanin we bought may not be identical to the indoline-betacyanin we synthesized, which suggests a deviation of the result from the graph.
Dopamine
The case for dopamine is slightly different. Since dopamine itself does not have color, we used hydrogen peroxide to oxidize dopamine into polydopamine and analyzed the absorbance spectrum of polydopamine (Wei, Zhang, Li, Li & Zhao, 2010; Zhu et al., 2018). We mixed 900 μL dopamine hydrochloride solution of different concentrations with 100 μL hydrogen peroxide, waited for 30 min, and measured the absorbance of the resulting polymer solution.
Polydopamine absorbance peaked at 371 nm. When we measured its absorbance at 371 nm, it showed a clear polynomial relationship with dopamine hydrochloride concentration.
Figure 4. Standard curve of absorbance of polydopamine, after oxidant-polymerization by hydrogen peroxide (H2O2). The results show a clear polynomial relationship between the starting concentration (before addition of H2O2) and absorbance of polydopamine. This polynomial relationship can be explained by the property of dopamine polymerization and the nature of absorbance.
Production of pigments and pigment precursor dopamine
Melanin
Melanin can be produced via the enzyme tyrosinase. With catalyzation of tyrosinase, L-Tyrosine is oxidized to L-DOPA and dopaquinone, which then undergoes spontaneous cyclization into eumelanin (Wang et al., 2019).
Figure 5. The metabolic pathway of melanin.
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℃.
Figure 6. (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℃.
Indigo
Indigo can be oxidized from L-Tryptophan by enzymes tryptophanase and FMO (Choi et al., 2003; iGEM19_GreatBay_SZ).
Figure 7. The metabolic pathway of indigo.
We constructed the plasmid J23102-TnaA-FMO. When trying to express the pigment in E. coli BL21(DE3) and V. natriegens, however, the bacterial solution turned orange. We hypothesized that indigo was reduced into leucoindigo (Roessler, Crettenand, Dossenbach, Marte & Rys, 2002), but the exact reason could not be found. SDS-PAGE protein electrophoresis showed expected results in terms of the length of enzymes.
Then, we tried to express the enzymes in another bacterial strain: E. coli DH5α. This time around, the bacterial solution turned blue. We added 100 mg/L L-Tryptophan in one shake flask and no substrate in another. Both shake flasks successfully produced indigo, and the one with the substrate produced more pigments than the other. The production in the bacteria with substrate is estimated to be 41.6 g/L.
Figure 8. Production of indigo in E. coli DH5α, with and without substrate, at 37℃ in 72 h.
Betalains
Out of the many types of betalains, we selected dopaxanthin (yellow) and indoline-betacyanin (pink-reddish) based on color and substrates needed. Betalains are produced from L-DOPA. 4,5-DOPA-extradiol dioxygenase oxidizes L-DOPA to 4,5-seco-DOPA, which then undergoes spontaneous cyclization to betalamic acid. Betalamic acid spontaneously combines with other substrates (amines or amino acids for betaxanthins, indoline derivatives for betacyanins, and L-DOPA for dopaxanthin) (Guerrero‐Rubio, López‐Llorca, Henarejos‐Escudero, García‐Carmona & Gandía‐Herrero, 2019).
Figure 9. The metabolic pathway of dopaxanthin and indoline-betacyanin.
We constructed the plasmid J23102-4,5-DODA and transformed the plasmid into E. coli BL21(DE3) and Vibrio natriegens. When we first tried to produce dopaxanthin and indoline-betacyanin in normal conditions (37℃, 220 rpm), the bacterial solution turned black. It is hypothesized that the pigments oxidized.
Next, we constructed the plasmid T7-4,5-DODA using Gibson Assembly. We added 0.1 mM IPTG for induction. After 20 h culture at 37℃, 220 rpm, we centrifuged the cells, discarded the LB medium, and resuspended the cell pellet in sterilized water. We then cultured the cells at 20℃, 120 rpm for 102 h and acquired expected results. We did not test the production in Vibrio natriegens because the bacterial strain we acquired (ATCC 14048) does not have the T7 promoter.
Figure 10. Production of dopaxanthin and indoline-betacyanin in E. coli BL21(DE3) after induction. (A) Production of dopaxanthin, after resuspension in sterilized water at 20℃, 120 rpm in 102 h. The horizontal axis is time (hours), and the vertical axis is absorbance of the bacterial solution at 415 nm. (B) Production of indoline-betacyanin, after resuspension in sterilized water at 20℃, 120 rpm in 102 h. The horizontal axis is time (hours), and the vertical axis is absorbance of the bacterial solution at 525 nm. (C) Production of dopaxanthin from 0-102 h. (D) Production of indoline-betacyanin from 0-102 h.
Dopamine
Dopamine can be oxidized from L-Tyrosine with HpaB, HpaC, and DDC enzymes (Das, Verma & Mukherjee, 2017).
Figure 11. The metabolic pathway of dopamine.
We successfully constructed the HpaBC-DDC plasmid and transformed it into E. coli BL21(DE3) and Vibrio natriegens. Expression of the enzymes HpaB, HpaC, and DDC were confirmed by an SDS-PAGE protein electrophoresis. However, the dopamine produced was often oxidized to polydopamine. We tried to produce dopamine in different conditions but failed in all of them.
Figure 12. Production of dopamine in E. coli BL21(DE3) and Vibrio natriegens at 25℃ and 37℃ 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℃.
Detecting the products of interest and quantifying production with HPLC-MS
We were honored to have the opportunity to use an HPLC-MS machine from Shimadzu Enterprise Management (China) Co., Ltd. to quantify the levels of production of indigo.
We first prepared indigo solutions of different concentrations to acquire a standard curve. Out of solutions of seven concentrations from 1-400 ppb, we acquired a linear standard curve of R^2=0.9955. We then diluted our product and measured its concentration. The concentration of our indigo was 880 μg/mL.
Figure 13. Measurement of the indigo standard curve (A) and production level (B) with HPLC-MS.
We also measured the accurate molecular weights of dopamine, dopaxanthin, and indoline-betacyanin and determined that we had produced the correct products. We were not able to quantify the melanin bioproduction because melanin is a polymer.
Figure 14. Mass spectrum results of dopamine, dopaxanthin, and indoline-betacyanin.
Hair bleach and hair dye
Hair bleach
Some of the hair we used were cut off from PIs or team members. To bleach the black hair, we mixed hydrogen peroxide milk with bleach powder at a ratio of 1:1. We let the hair stand for 45 min in the milk and successfully bleached the hair.
Figure 15. Hair before and after bleaching.
Hair dye
We first tried to dye hair using standard pigments. For dopamine, 10 mM CuSO4, a certain amount of H2O2, and ddH2O were mixed. 5 mg/mL standard dopamine hydrochloride was added. We incubated the mixture at 37℃ for 2 h. For melanin and indigo, we first incubated the hair in pH=9 Ca(OH)2 at 50℃ for 40 min. Next, we added 5 or 10 mg/mL pigment and let stand at 50℃ for another 40 min. For betacyanins, Ca(OH)2 was replaced by pH=5 sodium acetate (CH3COONa) buffer since the pigment shows color in acid solution (Reshmi, Aravindhan & Devi, 2012).
Figure 16. Hair dye results with standard pigments. (A) Hair dye results with standard indigo and melanin. (B) Hair dye results with standard betacyanins. (C) Hair dye results with hydrochloride dopamine combined with hydrogen peroxide.
Next, after we successfully produced the pigments, we attempted dyeing hair with our synthesized pigments using the same protocol. Although dopamine seemed to have been oxidized (when H2O2 was added, its color did not darken), we tried dyeing hair with the bacterial solution using the same method as the other pigments. The hair with synthesized melanin and indoline-betacyanin were dyed successfully, and the hair with indigo was partly dyed. Notably, the hair color dyed with synthesized indoline-betacyanin was darker than that dyed with standard betacyanins. It is hypothesized that indigo failed because the concentration was too low, dopaxanthin failed because the color of the bleached hair was too similar to dopaxanthin, and dopamine failed because most molecules were polydopamine, which was too large to get into the hair cortex.
Figure 17. Hair dye results with synthesized pigments, with positive and blank controls.
Expression and purification of bacterial laccase
Inspired by the synthetic dyes, which combine pigment precursors with oxidants, we thought of combining pigment precursors with an oxidase, laccase.
We constructed T7-laccase using the gene coding for laccase in Bacillus sp. (Mohammadian, Fathi-Roudsari, Mollania, Badoei-Dalfard & Khajeh, 2010). We successfully transformed the plasmid into E. coli BL21(DE3). We harvested the cells 20 h after IPTG induction and performed SDS-PAGE protein electrophoresis and an enzyme activity assay. Both tests showed the expected results.
Figure 18. (A) Verification of laccase using SDS-PAGE protein electrophoresis. (B) Laccase activity assay results.
Discussion
We have successfully expressed the corresponding enzymes and produced the pigments of interest. We additionally succeeded in dyeing hair with melanin, indigo, and indoline-betacyanin. These results lay the foundation of a method to mass-produce natural, harmless hair dyes, and these potential products shall bring great changes to the industry and our daily life.
We will need to optimize the dyeing methods for indigo and dopaxanthin (or other betaxanthins). For example, we may try to concentrate the pigments, raise the dyeing temperature, or change the method of opening the cuticles. We will also test if these pigments fade or get washed off easily. Furthermore, we shall design experiments to see whether these potential products are safe and whether they hurt the hair in any way. Finally, we may be able to apply for a patent or even eventually make it a product.
References
1. Wei, Q., Zhang, F., Li, J., Li, B., & Zhao, C. (2010). Oxidant-induced dopamine polymerization for multifunctional coatings. Polymer Chemistry, 1(9), 1430. doi: 10.1039/c0py00215a
2. Wang, Z., Tschirhart, T., Schultzhaus, Z., Kelly, E., Chen, A., & Oh, E. et al. (2019). Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application. Applied And Environmental Microbiology, 86(5). doi: 10.1128/aem.02749-19
3. Choi, H., Kim, J., Cho, E., Kim, Y., Kim, J., & Kim, S. (2003). A novel flavin-containing monooxygenase from Methylophaga sp. strain SK1 and its indigo synthesis in Escherichia coli. Biochemical And Biophysical Research Communications, 306(4), 930-936. doi: 10.1016/s0006-291x(03)01087-8
4. Team:GreatBay SZ/Composite Part - 2019.igem.org. (2020). Retrieved 17 October 2020, from https://2019.igem.org/Team:GreatBay_SZ/Composite_Part
5. Guerrero‐Rubio, M., López‐Llorca, R., Henarejos‐Escudero, P., García‐Carmona, F., & Gandía‐Herrero, F. (2019). Scaled‐up biotechnological production of individual betalains in a microbial system. Microbial Biotechnology, 12(5), 993-1002. doi: 10.1111/1751-7915.13452
6. Das, A., Verma, A., & Mukherjee, K. (2017). Synthesis of dopamine in E. coli using plasmid-based expression system and its marked effect on host growth profiles. Preparative Biochemistry & Biotechnology, 47(8), 754-760. doi: 10.1080/10826068.2017.1320291
7. Zhu, J., Tsehaye, M., Wang, J., Uliana, A., Tian, M., & Yuan, S. et al. (2018). A rapid deposition of polydopamine coatings induced by iron (III) chloride/hydrogen peroxide for loose nanofiltration. Journal Of Colloid And Interface Science, 523, 86-97. doi: 10.1016/j.jcis.2018.03.072
8. Roessler, A., Crettenand, D., Dossenbach, O., Marte, W., & Rys, P. (2002). Direct electrochemical reduction of indigo. Electrochimica Acta, 47(12), 1989-1995. doi: 10.1016/s0013-4686(02)00028-2
9. Mohammadian, M., Fathi-Roudsari, M., Mollania, N., Badoei-Dalfard, A., & Khajeh, K. (2010). Enhanced expression of a recombinant bacterial laccase at low temperature and microaerobic conditions: purification and biochemical characterization. Journal Of Industrial Microbiology & Biotechnology, 37(8), 863-869. doi: 10.1007/s10295-010-0734-5
10. Reshmi, S. K., Aravindhan, K. M., & Devi, P. S. (2012). The effect of light, temperature, pH on stability of betacyanin pigments in Basella alba fruit. Asian Journal of Pharmaceutical and Clinical Research, 5(4), 107-110.