Team:Shanghai SFLS SPBS/Engineering
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 section. For more specific characterization of the parts, please refer to the part collection.
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℃.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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