iGEM 2020 — Team Shanghai_SFLS_SPBS

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Shanghai SFLS SPBS/Notebook

* [Home](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS)
* Team
  + [Team members](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Team)
  + [Collaborations](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Partnership)
  + [Attributions](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Attributions)
* Project
  + [Description](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Description)
  + [Design](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Design)
  + [Experiments](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Notebook)
  + [Contribution](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Contribution)
  + [Results](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Results)
  + [Engineering](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Engineering)
  + [Implementation](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Proof_Of_Concept)
  + [Model](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Model)
* [Parts](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Parts)
* [Safety](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Safety)
* Human Practices
  + [Human Practices](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Human_Practices)
  + [Education & Public Engagement](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Education)
  + [Business Plan](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Entrepreneurship)
* [Poster](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Poster)

Document

![](https://static.igem.org/mediawiki/2020/c/c9/T--Shanghai_SFLS_SPBS--banner--2.4.png)

![](https://static.igem.org/mediawiki/2020/1/10/T--Shanghai_SFLS_SPBS--footer.png)

## We thank our sponsors

![](https://static.igem.org/mediawiki/2020/9/91/T--Shanghai_SFLS_SPBS--footer--1.png)
![](https://static.igem.org/mediawiki/2020/5/51/T--Shanghai_SFLS_SPBS--footer--2.png)
![](https://static.igem.org/mediawiki/2020/3/36/T--Shanghai_SFLS_SPBS--footer--3.png)
![](https://static.igem.org/mediawiki/2020/b/b0/T--Shanghai_SFLS_SPBS--footer--7.png)
![](https://static.igem.org/mediawiki/2020/c/cc/T--Shanghai_SFLS_SPBS--footer--4.png)
![](https://static.igem.org/mediawiki/2020/0/04/T--Shanghai_SFLS_SPBS--footer--5.png)
![](https://static.igem.org/mediawiki/2020/5/5c/T--Shanghai_SFLS_SPBS--footer--6.png)

## Contact us

Email: igem\_modu@126.com

WeChat public account: iGEM2020Modu

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Shanghai SFLS SPBS/Engineering

* [Home](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS)
* Team
  + [Team members](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Team)
  + [Collaborations](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Partnership)
  + [Attributions](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Attributions)
* Project
  + [Description](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Description)
  + [Design](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Design)
  + [Experiments](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Notebook)
  + [Contribution](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Contribution)
  + [Results](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Results)
  + [Engineering](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Engineering)
  + [Implementation](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Proof_Of_Concept)
  + [Model](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Model)
* [Parts](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Parts)
* [Safety](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Safety)
* Human Practices
  + [Human Practices](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Human_Practices)
  + [Education & Public Engagement](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Education)
  + [Business Plan](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Entrepreneurship)
* [Poster](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Poster)

Document

![](https://static.igem.org/mediawiki/2020/b/b1/T--Shanghai_SFLS_SPBS--banner--2.7.png)

We designed the parts J23102-Tyr1, J23102-TnaA-FMO, J23102-4,5-DODA, J23102-HpaBC-DDC, T7-laccase, and pTac-RBS-Tyr1 in various strains of bacteria. We used them to produce pigments, pigment precursors, and bacterial laccase. We then used the pigments or combined pigment precursors with laccase to dye hair.

This page will document the experimenting process of these parts. An overview of our experiments can be seen in the [results](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Results) section. For more specific characterization of the parts, please refer to the [part collection](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Parts).

# Melanin production (Tyr1)

We first attempted expressing Tyr1 with a constitutive promoter, J23102, and successfully produced melanin. We then used Gibson Assembly to substitute J23102 with pTac and tried to express Tyr1 with IPTG induction.

## Expression and production using the constitutive 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℃. ![](https://static.igem.org/mediawiki/2020/e/ed/T--Shanghai_SFLS_SPBS--melanin.png) Figure 1. (A) Production of melanin in E. coli BL21(DE3) and Vibrio natriegens at 25℃ and 37℃ in 72 h. The horizontal axis is time (hours), and the vertical axis is the absorbance of the bacterial solution at 400 nm (1:10 diluted if necessary). (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℃. Expression and production with inductive promoter pTac We tried to express tyrosinase (Tyr1) with the inductive promoter pTac. Although melanin was produced, the rate of production was much lower than that with the constitutive promoter J23102. ![](https://static.igem.org/mediawiki/2020/0/02/T--Shanghai_SFLS_SPBS--pTac-Tyr1.png) Figure 2. Production of melanin in E. coli BL21(DE3) and Vibrio natriegens with pTac-Tyr1. Indigo production (TnaA-FMO) We first tried to produce indigo with E. coli BL21(DE3) and Vibrio natriegens ATCC 14048. However, although SDS-PAGE protein electrophoresis showed enzymes of the correct lengths, the bacterial solution turned orange instead of blue. We retried production in E. coli DH5α and obtained success. Failed production in E. coli BL21(DE3) and Vibrio natriegens ATCC 14048 We constructed the plasmid J23102-TnaA-FMO. When trying to express the pigment in E. coli BL21(DE3) and V. natriegens at 25℃ and 37℃, the bacterial solution turned orange, instead of the expected blue. SDS-PAGE protein electrophoresis showed enzymes of the correct length. ![](https://static.igem.org/mediawiki/2020/3/3d/T--Shanghai_SFLS_SPBS--picture--2.7.2.jpg) Figure 3. Initial results of TnaA-FMO. Top-left: E. coli BL21(DE3) at 25℃. Top-right: E. coli BL21(DE3) at 37℃. Bottom-left: V. natriegens at 25℃. Bottom-right: V. natriegens at 37℃. We first doubted that the design of our plasmids were incorrect. Having learned that GreatBay\_SZ from iGEM 2019 successfully produced indigo, we borrowed some preserved bacteria from them, extracted the plasmids, transformed them into E. coli BL21(DE3) and V. natriegens, and carried out another round of experiment. Nevertheless, this time around, the bacterial solution still turned orange. After research, we hypothesized that indigo was reduced into leucoindigo (Roessler, Crettenand, Dossenbach, Marte & Rys, 2002). Some reducing agents may exist in E. coli BL21(DE3) and V. natriegens and prevent us from acquiring the correct pigments. Successful production in E. coli DH5α We decided to try producing indigo in E. coli DH5α, the bacterial strain used by iGEM19\_GreatBay\_SZ. Surprisingly (frankly speaking, not so surprising), the tubes showed blue color. We then used shake flasks and E. coli DH5α to produce indigo. 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 flask with substrates is estimated to be 41.6 g/L. ![](https://static.igem.org/mediawiki/2020/c/cf/T--Shanghai_SFLS_SPBS--indigo.png) Figure 4. (A) Production of indigo in E. coli DH5α, with and without substrate, at 37℃ in 72 h. (B) Production of indigo from 0-60 h. Betalains production (4,5-DODA) Based on substrate availability and color, we chose to express dopaxanthin and indoline-betacyanin. We first attempted expressing 4,5-DODA and producing the betalains at 37℃, 220 rpm with the constitutive promoter J23102. However, the bacterial solution turned black. It was hypothesized that the pigments had oxidized. We retried production with promoter J23102 at low temperature, low oxygen level (20℃, 120 rpm) but once again did not get the expected results. Next, we used Gibson Assembly to replace J23102 with T7-LacO. We tried to produce the two betalains for the third time and were able to acquire the pigments. Failed production at normal temperature and oxygen levels 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. ![](https://static.igem.org/mediawiki/2020/8/8e/T--Shanghai_SFLS_SPBS--picture--2.7.4.png) Figure 5. Initial production results of dopaxanthin (A) and indoline-betacyanin (B). Top-left: E. coli BL21(DE3) at 25℃. For each picture, the bacteria and temperature conditions are as follows. Top-right: E. coli BL21(DE3) at 37℃. Bottom-left: V. natriegens at 25℃. Bottom-right: V. natriegens at 37℃. Failed production at low temperature, low oxygen level We learned from works of literature that betalains should be produced at a low temperature and with low oxygen levels (Guerrero‐Rubio, López‐Llorca, Henarejos‐Escudero, García‐Carmona & Gandía‐Herrero, 2019). 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. However, the experiment failed again, and the bacterial solutions did not change significantly in color or absorbance at the expected wavelengths. Successful production with inductive promoter T7 in E. coli BL21(DE3) We constructed the plasmid T7-4,5-DODA using Gibson Assembly. We added 0.1 mM IPTG for induction. Again, 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. ![](https://static.igem.org/mediawiki/2020/4/48/T--Shanghai_SFLS_SPBS--picture--2.6.10.png) Figure 6. 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 the 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 the 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 production (HpaBC-DDC) 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 was confirmed by SDS-PAGE protein electrophoresis. However, the dopamine produced was often oxidized to polydopamine. We tried to produce dopamine in different conditions, including at different temperatures and different oxygen levels, but all of them failed. We learned that dopamine can polymerize in acidic, basic, as well as high-oxygen conditions (Du et al., 2014; Du et al., 2017; Chen, Liu, Su & Liang, 2017; Wei, Zhang, Li, Li & Zhao, 2010). ![](https://static.igem.org/mediawiki/2020/9/94/T--Shanghai_SFLS_SPBS--picture--2.6.12.jpg) Figure 7. Production of dopamine in E. coli BL21(DE3) and Vibrio natriegens at 25℃ and 37℃ in 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. ![](https://static.igem.org/mediawiki/2020/3/3d/T--Shanghai_SFLS_SPBS--picture--2.6.13.png) Figure 8. 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. ![](https://static.igem.org/mediawiki/2020/a/a4/T--Shanghai_SFLS_SPBS--HPLC-MS-small.png) Figure 9. Mass spectrum results of dopamine, dopaxanthin, and indoline-betacyanin. Bacterial laccase expression and purification 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. 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. ![](https://static.igem.org/mediawiki/2020/e/ec/T--Shanghai_SFLS_SPBS--picture--2.6.laccase.png) Figure 10. (A) Verification of laccase using SDS-PAGE protein electrophoresis. (B) Laccase activity assay results. References 1.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 2.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 3.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 4.Du, X., Li, L., Li, J., Yang, C., Frenkel, N., & Welle, A. et al. (2014). UV-Triggered Dopamine Polymerization: Control of Polymerization, Surface Coating, and Photopatterning. Advanced Materials, 26(47), 8029-8033. doi: 10.1002/adma.201403709 5.Du, X., Li, L., Behboodi-Sadabad, F., Welle, A., Li, J., & Heissler, S. et al. (2017). Bio-inspired strategy for controlled dopamine polymerization in basic solutions. Polymer Chemistry, 8(14), 2145-2151. doi: 10.1039/c7py00051k 6.Chen, T., Liu, T., Su, T., & Liang, J. (2017). Self-Polymerization of Dopamine in Acidic Environments without Oxygen. Langmuir, 33(23), 5863-5871. doi: 10.1021/acs.langmuir.7b01127 7.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

![](https://static.igem.org/mediawiki/2020/1/10/T--Shanghai_SFLS_SPBS--footer.png)

## We thank our sponsors

![](https://static.igem.org/mediawiki/2020/9/91/T--Shanghai_SFLS_SPBS--footer--1.png)
![](https://static.igem.org/mediawiki/2020/5/51/T--Shanghai_SFLS_SPBS--footer--2.png)
![](https://static.igem.org/mediawiki/2020/3/36/T--Shanghai_SFLS_SPBS--footer--3.png)
![](https://static.igem.org/mediawiki/2020/b/b0/T--Shanghai_SFLS_SPBS--footer--7.png)
![](https://static.igem.org/mediawiki/2020/c/cc/T--Shanghai_SFLS_SPBS--footer--4.png)
![](https://static.igem.org/mediawiki/2020/0/04/T--Shanghai_SFLS_SPBS--footer--5.png)
![](https://static.igem.org/mediawiki/2020/5/5c/T--Shanghai_SFLS_SPBS--footer--6.png)

## Contact us

Email: igem\_modu@126.com

WeChat public account: iGEM2020Modu

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Shanghai SFLS SPBS/Experiments

* [Home](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS)
* Team
  + [Team members](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Team)
  + [Collaborations](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Partnership)
  + [Attributions](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Attributions)
* Project
  + [Description](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Description)
  + [Design](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Design)
  + [Experiments](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Notebook)
  + [Contribution](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Contribution)
  + [Results](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Results)
  + [Engineering](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Engineering)
  + [Implementation](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Proof_Of_Concept)
  + [Model](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Model)
* [Parts](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Parts)
* [Safety](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Safety)
* Human Practices
  + [Human Practices](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Human_Practices)
  + [Education & Public Engagement](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Education)
  + [Business Plan](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Entrepreneurship)
* [Poster](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Poster)

Document

![](https://static.igem.org/mediawiki/2020/2/25/T--Shanghai_SFLS_SPBS--banner--2.3.png)

# Culture medium preparation

The following is the recipe for 1 L culture medium. For solid plates, 10 g/L agar was added.

## LB Medium

|  |  |
| --- | --- |
| Tryptone | 10.0 g/L |
| Yeast Extract | 5.0 g/L |
| NaCl | 10.0 g/L |
| ddH2O | To 1 L |

## LBv2 Medium

|  |  |
| --- | --- |
| Tryptone | 10.0 g/L |
| Yeast Extract | 5.0 g/L |
| NaCl | 21.9 g/L |
| KCl | 0.31 g/L |
| MgCl2·6H2O | 4.70 g/L |
| ddH2O | To 1 L |

## M9 Medium

|  |  |
| --- | --- |
| M9 minimal salts 5x | 11.28 g/L |
| glucose | 4 g/L |
| NaCl | 0.5 g/L |
| CaCl2 | 10 mg/L |
| MgSO4 | 0.5 g/L |
| CuSO4 | 40 mg/L |
| ddH2O | To 1 L |

# Strain inoculation

For solid plates, a pipette tip or toothpick was used to pick a single colony from the plate and put into the culture medium. Bacterial solution was added 1:100 to the culture medium.

# Strain preservation

After overnight culture, 500 μL (or 800 μL) bacterial solution was mixed with 500 μL (or 800 μL) 30% glycerol (sterilized) in a EP tube. The tube was preserved at the -80°C refrigerator.

# Chemical transformation for E. coli

1. 50 μL of E. coli competent cells and 0.5-1 μL plasmid (2 μL for Gibson Assembly products) was mixed on ice. The tube was placed on ice bath for 20 min.

2. The tube was subjected to a heat shock at 42℃ for 60 s.

3. The tube was ice bathed for 2 min.

4. 300 μL LB culture medium without antibiotics was added, and the bacteria were cultured in a shaker at 37℃ for 60 min.

5. For plasmid transformation, 100 μL of bacterial solution was taken and spread on a solid plate containing antibiotics. The plates were cultured at 37℃ overnight. For the transformation of Gibson Assembly products, the bacterial solution was centrifuged at 4000 rpm for 1 min. 250 μL supernatant was removed and the bacterial pellet was resuspended with remaining LB medium. The resuspension was then inoculated on a solid plate containing antibiotics and incubated at 37℃ overnight.

# V. natriegens competent cell preparation

1. V. Natriegens cells were grown in LBv2 medium overnight at 37℃, 220 rpm.

2. The cultures from Step 1 were used to inoculate (1:200 dilution) a second matched medium culture (100 mL), which was grown at 37℃, 220 rpm until an OD600 of 1.0.

3. The culture was split into centrifuge bottles and pelleted at 6465 g for 10 min at room temperature.

4. The supernatant was decanted, and the cell pellets were gently resuspended in 3.25 mL of prewarmed 1 M sorbitol (sterilized) and incubated at 37℃ for 1 h.

# Chemical transformation for V. natriegens

1. Plasmid DNA (typically 1-2 μL of 100 ng/μL) and 200 μL chemically competent cells (in 1 M sorbitol) were combined and gently mixed on ice.

2. The mixture was transferred to a heat bath (42℃) and subjected to heat shock for 2.5 min.

3. The cells were immediately recovered in 500 μL of recovery medium (LBv2 substituted with 680 mM sucrose, sterilized) and transferred to a 15 mL conical tube.

4. Cells were incubated at 37℃, 220 rpm overnight.

5. 150 μL aliquots of the recovery medium were plated on warm agar plates containing the appropriate antibiotic.

6. The plates were incubated at 37℃ overnight (~10-12 h) for colony growth.

# Plasmid extraction

TIANprep Mini Plasmid Kit (DP103 from Tiangen Biotech (Beijing) Co., Ltd.) was used for plasmid extraction.

1. A new Spin Column CP3 was placed in the collection tube. 500 μL Buffer BL was added to the column. The collection tube was centrifuged at 12,000 rpm for 1 min, and waste liquid was discarded.

2. 1-5 ml of overnight cultured bacteria solution was added to a centrifuge tube and centrifuge for 1 min at 12,000 rpm.

3. The supernatant was removed, and 250 μL Buffer P1 (with RNase A added) was added to the centrifuge tube with the bacterial pellet. The bacterial pellet was completely suspended.

4. 250 μL Buffer P2 was added to the centrifuge tube and the tube was gently inverted 6-8 times to fully lyse the cells.

5. 350 μL Buffer P3 was added to the centrifuge tube, which was immediately yet gently inverted 6-8 times. The system was centrifuged at 12,000 rpm for 10 min.

6. The supernatant collected in Step 5 was transferred to Spin Column CP3. The column was centrifuge at 12,000 rpm for 1 min and the waste was discarded.

7. 600 μL Buffer PW (with ethanol added) was added to the Spin Column CP3. The column was centrifuge at 12,000 rpm for 1 min and the waste was discarded.

8. Step 7 was repeated.

9. The column was placed in the collection tube and centrifuge at 12,000 rpm for 2 min to remove the residual Buffer PW.

10. The Spin Column CP3 was placed in a clean EP tube. 50-100 μL ddH2O was added. The column was placed at room temperature for 2 min, then centrifuged at 12,000 rpm for 2 min.

11. The purity and concentration of the extracted plasmids was measured.

# Colony PCR

2x Taq PCR MasterMix (KT201 from Tiangen Biotech (Beijing) Co., Ltd.) was used for plasmid extraction.

1. The following reaction system was prepared. The system was gently mixed

|  |  |
| --- | --- |
| 2 x Taq PCR Mix | 10 μL |
| Forward Primer（10 μM） | 1 μL |
| Reverse Primer（10 μM） | 1 μL |
| Colony | 1 single colony (1 μL bacterial solution) |
| ddH2O | to 20 μL |

2. The PCR program was set as follows:

| Step | Temperature | Time | Cycle |
| --- | --- | --- | --- |
| Initial denaturation | 94℃ | 10 min | 1 cycle |
| Denaturation | 94℃ | 30 s | 30 cycles |
| Annealing | (Tm)-5~10℃ | 30 s |
| Extension | 72℃ | 1 kb/min |
| Final extension | 72℃ | 5 min | 1 cycle |
| Hold | ≤16℃ | ∞ | - |

# Gibson Assembly

Golden Star T6 Super PCR Mix (from Beijing Tsingke Biotech Co., Ltd.) was used for the reaction.

1. The following reaction system was configured on ice.

|  |  |
| --- | --- |
| Golden Star T6 Super PCR Mix | 47 μL |
| Plasimd with interested gene | 1 μL |
| Forward primer | 1 μL |
| Reverse primer | 1 μL |

2. A pipette was used to gently mix the system.

3. A PCR was first carried out for the system. The program was set as follows:

| Step | Temperature | Time | Cycle |
| --- | --- | --- | --- |
| Initial denaturation | 98℃ | 2 min | 1 cycle |
| Denaturation | 98℃ | 10 s | 30 cycles |
| Annealing | (Tm)-5~10℃ | 10 s |
| Extension | 72℃ | ~30 s/kb |
| Final extension | 72℃ | 5 min | 1 cycle |
| Hold | 16℃ | ∞ | - |

4. After the PCR, the vector and the fragment were mixed. The reaction tubes was placed in the PCR machine and incubated at 50℃ for 15 min.

# Agarose gel electrophoresis

1. 0.25 g/0.5g/1.0g agarose and 25 mL/50mL/100mL of 1×TAE buffer was mixed in a flask. The solution was heated in a microwave oven until agarose dissolves completely.

2. 2.5 μL/5 μL/10 μL SYBR safe DNA Gel Stain (10000×) was added. After mixing the solution, the agarose was poured into a gel tray with the well comb in place. The gel was left for 20-30 min to completely solidify.

3. After the liquid solidifies, the well comb was unplugged.

4. The gel was put into the electrophoresis unit. 1×TAE buffer was added to the unit until the gel was completely immersed in the buffer.

5. For PCR products with loading dye (e.g. those that used 2x Taq Mix), 3-6 μL DNA (PCR product) was loaded into each lane. For samples without loading dye, 4 μL DNA was mixed with 1 μL loading dye before being loaded into the lanes. One or multiple markers were loaded as needed.

6. The voltage was set to 120 V for 20-30 min.

# DNA purification

TIANgel Midi Purification Kit (DP209 from Tiangen Biotech (Beijing) Co., Ltd.) was used for DNA purification.

1. 500 μL Buffer BL was added into a Spin Column CA2, which was centrifuged at 12,000 rpm for 1 minute. Waste liquid was discarded from the collection tube.

2. As much agarose gel as possible was carefully removed from the target DNA strip. The DNA strip was placed in a clean centrifuge tube for weighing.

3. The same amount of Buffer PN was added. The mixture was placed in 50°C water bath for 10 min. The tube was gently turned to ensure that the gel was fully dissolved in the buffer.

4. The solution obtained from Step 3 was added into a Spin Column CA2 and left at room temperature for 2 min. The solution was centrifuged at 12,000 rpm for 1 min, and the waste liquid was discarded.

5. 600 μL Buffer PW (with ethanol added before use) was added to the column. The column was centrifuged at 12,000 rpm for 1 min. The waste liquid was discarded.

6. Step 5 was repeated.

7. The Spin Column CA2 was centrifuged at 12,000 rpm for another 2 min to remove any remaining buffer. It was then placed at room temperature for a few minutes to dry completely.

8. The column was moved onto a clean EP tube. 50-100 μL ddH2O was added. The column was place at room temperature for a few minutes and then centrifuge it at 12,000 rpm for 2 min.

9. The purity and concentration of the recovered DNA was measured and recorded.

# Protein electrophoresis

BeyoGel™ Plus Precast PAGE Gel (4-20%, 15 wells) from Beyotime Biotechnology was used as the SDS-PAGE gel for protein electrophoresis.

1. The precast gel was fixed in the electrophoresis tank. The comb was gently pulled out.

2. The electrophoresis buffer was prepared using SDS-PAGE Electrophoresis Buffer with Biofuraw™ Pre-Cassette Gel-Running Buffer.

3. The electrophoresis tank was filled with electrophoresis buffer. The buffer was poured into the inner tank until it overflowed to the outer tank and submerged the anode at the bottom of the tank.

4. 4-10 μL of each sample was gently loaded into one lane. One or multiple markers were loaded as needed.

5. The voltage was set to 120 V for 20-30 min.

6. The precast gel was taken out of the tank. A blade was used to separate the gel from the cast.

7. An appropriate amount of protein dye (Biofuraw™ LDS Sample Buffer, With Reducing, diluted from 4X) was added so that the staining solution could cover the gel and the liquid level is at least three times higher than the thickness of the gel. Dyeing was performed on a shaker at room temperature (20-25°C) for 20-30 min.

# Hair bleach

1. Bleach powder and hydrogen peroxide milk were mixed 1:1.

2. Black hair was added and let stand for 50 min.

# Hair dye

1. pH=9 Ca(OH)2 (for melanin, polydopamine, indigo, and dopaxanthin) or pH=5 sodium acetate buffer (for indoline-betacyanin and laccase+precursors) was prepared.

2. An appropriate amount of hair was added to the solution and let stand at 50℃ for 40 min to open the cortex of the hair.

3. The solution was discarded and 10 mg/mL pigment solution was added.

4. The system was let stand at 50℃ for another 40 min.

5. The hair was washed with excessive water.

# Production & measurement of pigments

Single colonies or preserved bacteria (1:100) was added to 5 mL culture medium (LB for E. coli, LBv2 for Vibrio natriegens) and cultured for approximately 12 h. Next, 500 μL bacterial solution was added to 50 mL matching culture medium, the appropriate substrates are added, and this time is recorded as 0 h.

# Melanin

1. 50 mg/L L-DOPA and 8 mg/L L-Tyrosine was added at 0 h. The bacteria were cultured at 37℃, 220 rpm.

2. Every 12 h, 2 mL bacterial solution was moved to a EP tube, the tube was centrifuged, and absorbance at 400 nm was measured and recorded.

3. The production curve was measured over 72 h.

# Indigo

1. 100 mg/L L-Tryptophan was added at 0 h. The bacteria were cultured at 37℃, 220 rpm.

2. Every 12 h, 2 mL bacterial solution was moved to a EP tube

3. The tube was centrifuged for 1 min, the supernatant was discarded, and 2 mL DMSO was added to resuspend the indigo.

4. The tube was centrifuged for another 1 min to make sure the bacteria is not in the solution, which may affect measurement of absorbance.

5. Absorbance at 605 nm was measured and recorded.

6. The production curve was measured over 72 h.

# Dopaxanthin & betacyanin (T7-4,5-DODA)

1. The bacterial strains with the inducible promoter was cultured at 37℃, 220 rpm until OD600=1.0.

2. 0.1 mM IPTG was added in both flasks. The flasks were cultured at 37℃, 220 rpm.

3. After 16 h, the bacterial solution was centrifuged at 5000 ×g for 10 min. The supernatant was discarded and the cell pellet was resuspended in 50 mL sterilized ddH2O.

4. In the flask for dopaxanthin, 9 g/L L-DOPA and 2.64 g/L Vitamin C (sodium L-ascorbate) was added. In the flask for indoline-betacyanin, 1.5 g/L L-DOPA, 0.0452 g/L indoline, and 2.64 g/L Vitamin C was added. The substrates were added at 0 h.

5. The flasks were cultured at 20℃, 120 rpm. Every 12 h, 2 mL bacterial solution was moved to a EP tube, the tube was centrifuged, and absorbance at 415 nm (dopaxanthin) and 525 nm (indoline-betacyanin) was measured and recorded.

6. The production curve was measured over 96 h.

# IPTG Induction

After overnight culture in shaking tubes, the bacterial solution was added 1:100 into a matching culture medium and cultured at 37℃, 220 rpm for ~3 h (or until OD600~1.0). For T7 promoters, 0.1 mM IPTG was added. For pTac promoters, 1 mM IPTG was added.

# References

1. Retrieved July 2020, from https://www.protocols.io/view/growth-media-for-v-natriegens-u3ceyiw

2. Retrieved July 2020, from http://cshprotocols.cshlp.org/content/2010/8/pdb.rec12295.short

3. T. Tschirhart et al., "Synthetic Biology Tools for the Fast-Growing Marine Bacterium Vibrio natriegens", ACS Synthetic Biology, vol. 8, no. 9, pp. 2069-2079, 2019. Available: 10.1021/acssynbio.9b00176.

![](https://static.igem.org/mediawiki/2020/1/10/T--Shanghai_SFLS_SPBS--footer.png)

## We thank our sponsors

![](https://static.igem.org/mediawiki/2020/9/91/T--Shanghai_SFLS_SPBS--footer--1.png)
![](https://static.igem.org/mediawiki/2020/5/51/T--Shanghai_SFLS_SPBS--footer--2.png)
![](https://static.igem.org/mediawiki/2020/3/36/T--Shanghai_SFLS_SPBS--footer--3.png)
![](https://static.igem.org/mediawiki/2020/b/b0/T--Shanghai_SFLS_SPBS--footer--7.png)
![](https://static.igem.org/mediawiki/2020/c/cc/T--Shanghai_SFLS_SPBS--footer--4.png)
![](https://static.igem.org/mediawiki/2020/0/04/T--Shanghai_SFLS_SPBS--footer--5.png)
![](https://static.igem.org/mediawiki/2020/5/5c/T--Shanghai_SFLS_SPBS--footer--6.png)

## Contact us

Email: igem\_modu@126.com

WeChat public account: iGEM2020Modu

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Shanghai SFLS SPBS/Results

* [Home](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS)
* Team
  + [Team members](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Team)
  + [Collaborations](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Partnership)
  + [Attributions](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Attributions)
* Project
  + [Description](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Description)
  + [Design](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Design)
  + [Experiments](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Notebook)
  + [Contribution](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Contribution)
  + [Results](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Results)
  + [Engineering](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Engineering)
  + [Implementation](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Proof_Of_Concept)
  + [Model](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Model)
* [Parts](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Parts)
* [Safety](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Safety)
* Human Practices
  + [Human Practices](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Human_Practices)
  + [Education & Public Engagement](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Education)
  + [Business Plan](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Entrepreneurship)
* [Poster](https://2020.igem.org/Team%3AShanghai_SFLS_SPBS/Poster)

Document

![](https://static.igem.org/mediawiki/2020/a/a4/T--Shanghai_SFLS_SPBS--banner--2.6.png)

# 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.

![](https://static.igem.org/mediawiki/2020/5/5b/T--Shanghai_SFLS_SPBS--picture--2.6.1.png)

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.

![](https://static.igem.org/mediawiki/2020/0/00/T--Shanghai_SFLS_SPBS--picture--2.6.2.png)

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.

![](https://static.igem.org/mediawiki/2020/0/0c/T--Shanghai_SFLS_SPBS--picture--2.6.3.png)

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.

![](https://static.igem.org/mediawiki/2020/2/2a/T--Shanghai_SFLS_SPBS--picture--2.6.4.png)

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).

![](https://static.igem.org/mediawiki/2020/9/98/T--Shanghai_SFLS_SPBS--picture--2.1.1.png)

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℃.

![](https://static.igem.org/mediawiki/2020/e/ed/T--Shanghai_SFLS_SPBS--melanin.png)

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).

![](https://static.igem.org/mediawiki/2020/d/d5/T--Shanghai_SFLS_SPBS--picture--2.1.3.png)

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.

![](https://static.igem.org/mediawiki/2020/c/cf/T--Shanghai_SFLS_SPBS--indigo.png)

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).

![](https://static.igem.org/mediawiki/2020/1/12/T--Shanghai_SFLS_SPBS--picture--2.1.4.png)

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.

![](https://static.igem.org/mediawiki/2020/4/48/T--Shanghai_SFLS_SPBS--picture--2.6.10.png)

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).

![](https://static.igem.org/mediawiki/2020/b/b8/T--Shanghai_SFLS_SPBS--picture--2.1.2.png)

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.

![](https://static.igem.org/mediawiki/2020/9/94/T--Shanghai_SFLS_SPBS--picture--2.6.12.jpg)

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.

![](https://static.igem.org/mediawiki/2020/3/3d/T--Shanghai_SFLS_SPBS--picture--2.6.13.png)

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.

![](https://static.igem.org/mediawiki/2020/a/a4/T--Shanghai_SFLS_SPBS--HPLC-MS-small.png)

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.

![](https://static.igem.org/mediawiki/2020/b/b9/T--Shanghai_SFLS_SPBS--picture--2.6.14.png)

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).

![](https://static.igem.org/mediawiki/2020/f/f7/T--Shanghai_SFLS_SPBS--picture--2.6.15.png)

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.

![](https://static.igem.org/mediawiki/2020/d/df/T--Shanghai_SFLS_SPBS--picture--2.6.16.png)

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.

![](https://static.igem.org/mediawiki/2020/e/ec/T--Shanghai_SFLS_SPBS--picture--2.6.laccase.png)

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.

![](https://static.igem.org/mediawiki/2020/1/10/T--Shanghai_SFLS_SPBS--footer.png)

## We thank our sponsors

![](https://static.igem.org/mediawiki/2020/9/91/T--Shanghai_SFLS_SPBS--footer--1.png)
![](https://static.igem.org/mediawiki/2020/5/51/T--Shanghai_SFLS_SPBS--footer--2.png)
![](https://static.igem.org/mediawiki/2020/3/36/T--Shanghai_SFLS_SPBS--footer--3.png)
![](https://static.igem.org/mediawiki/2020/b/b0/T--Shanghai_SFLS_SPBS--footer--7.png)
![](https://static.igem.org/mediawiki/2020/c/cc/T--Shanghai_SFLS_SPBS--footer--4.png)
![](https://static.igem.org/mediawiki/2020/0/04/T--Shanghai_SFLS_SPBS--footer--5.png)
![](https://static.igem.org/mediawiki/2020/5/5c/T--Shanghai_SFLS_SPBS--footer--6.png)

## Contact us

Email: igem\_modu@126.com

WeChat public account: iGEM2020Modu