Team:BJU China/Description
project Description
Inspiration
Purple originated in Byzantium, known as tyrian purple, or royal purple. It is one of the derivatives of Indigo dyes.
It is a dye extracted from Mediterranean sea snails. As far back as 2000 BC, people began to use see snails to produce tyrian purple for dyeing, based on the principle that the secretion of the snail is discolored by light. Although snail dyeing techniques had been continuously developed since then, there were merely two techniques: kill snails, cut out the subgill glands and let them expose to light; or pinch and stimulate glands to secrete dying substance. "Rare things are precious." Once discovered at a time when purple dyes were quite pre-existent, snail dyeing quickly became popular. As a result, it was highly appreciated by the upper social classes of the Roman Empire. Purple then became a symbol of royalty and power.
However, all things bright and luxurious come at prices. As we investigated further, we found that:
Dyes are responsible for up to one-fifth of the world's industrial water pollution. This sewage is usually discharged directly into rivers and streams. Emissions are often a mixture of carcinogenic chemicals, or the mixture of dyes, salts and heavy metals. This is a serious problem because the chemicals kill plants, animals and even threaten human health. Let's take one of the dyes, indigo, for example. In India, Indigo effluent has caused a number of cases of drinking water poisoning. In China, over 70% of the rivers and 90 percent of the local groundwater are polluted. Toxic chemicals are so hard to decompose that it would exist in water sources for a long period of time.
Project Goal
Our goal was clear - to create an environmental-friendly indigo dye with less pollution. We intended to produce this dye biosynthetically: we cultured and produced the indigo dye and its derivatives, especially tyrian purple, in E. coli.
The main challenge of this production is that it is difficult to introduce two Br atoms into tyrian purple. To resolve this difficulty, through researches, two main methods were found to introduce Br atoms: Most methods use bromine gas or hydrogen bromide for bromination, but they lack regional specificity, low product yield, and harmful to the environment in the manufacturing processes; The high cost of brominated precursors prevents the synthesis of 6BrlG from brominated precursors.
To solve the difficulty of region-specific bromination, we used new biocatalytic techniques. One of the most reliable biocatalysts is Tryptophan halogenase because of its high regional specificity. Tryptophan is halogenated with halide ions (X-) dependent on the Tryptophan halogenase (Flavin). Here, we selected Trp 6-halogenase(Stth) from S. oxytricini to brominate Trp to 6-br-TrP in E.coli.
However, previous studies have shown that the enzyme has a low solubility when expressed in E. coli. Therefore, the soluble protein Flavin reductase (Fre) in E. coli was connected to the N-terminal of Stth, and fusion enzyme L3 (Linker sequence: AEAAAKEAAAKA) was used for connection.
Next, we converted 6-Br-Trp into 6-Br-Indole, and as the substrate, indigo substances were generated under the action of oxygenases, thus producing various colored indigo dyes. After reviewing the literature, we used TnaA to convert 6-Br-Trp to 6-Br-Indole and oxygenase MaFMO to convert 6-Br-Trp to 6,6'-dibromoindigo(6BrIG).
These two processes were carried out in E. coli BL21 ΔTnaA strain respectively，and then integrated through two-cell system. It can be summarized as follows:
In order to expand the applicability of our dyes, we found that we could produce different halogenated indigos by changing the fusion halogenase (Fre-L3-SttH) and the salt (sodium bromide) used in the production of aurochromis. The rest steps remain unchanged, and the product results are shown in the figure below:
Future Application
Indigo supports reversible dielectron reduction and dielectron oxidation. They have good charge transport mobility and are a unique and malleable pigment. It is biodegradable and non-toxic as well. Another important feature is the typically high environmental stability and their ability to form hydrogen bonds. In contrast to van der Waals interactions, a hydrogen bond is a very strong intermolecular interaction. In addition, studies have found that the ability of indigo to conduct electricity is affected by light. When exposed to light, indigo substances will change from the ground state S0 to the first excited state S1. After absorbing the energy of light, the hydrogen atoms on NH will be transferred from the ketone form to the enol form (as shown in figure A below), thus possessing the ability to transmit charge. All this gives indigo the properties of photosensitive semiconductors, especially dye-sensitive cells.
Furthermore, we will combine tyrian purple with art. Synthetic biology and art, in most people's minds, may be like the earth's north and south poles: synthetic biology aims to engineer life, while art is to show the most essential meaning. In our project, we used synthetic biology to redefine art. We used pigments produced from E.coli to express the artist's thoughts, to involve the artists in the transformation of living things, and to give art a new meaning.
Art is unconstrained; Synthetic biology is rigorous. The combination of biology and art is not only breaking historical conventions, but also innovative.
Reference
Cammarota, K. (2019, April 12). Indigo dye is affecting the water supply in India. Retrieved August 3, 2021, from https://borgenproject.org/indigo-dye-is-affecting-the-water-supply-in-india/
Głowacki, E. D., Voss, G., & Sariciftci, N. S. (2013). 25th anniversary article: progress in chemistry and applications of functional indigos for organic electronics. Advanced materials (Deerfield Beach, Fla.), 25(47), 6783–6800. https://doi.org/10.1002/adma.201302652
Harvay, L. (2017, March 20). Purple owes its association with royalty to the high expense of creating the dye. Vintage News. Retrieved September 11, 2021, from https://www.thevintagenews.com/2017/03/20/purple-owes-its-association-with-royalty-to-the-high-expense-of-creating-the-dye/
Irwin, M. D., Lovelace, J., & Mielczarek, K. (2021, July 28). System and method for testing photosensitive device degradation.
Klimovich, I. V., Leshanskaya, L. I., Troyanov, S. I., Anokhin, D. V., Novikov, D. V., Piryazev, A. A., Ivanov, D. A., Dremova, N. N., & Troshin, P. A. (2014). Design of indigo derivatives as environment-friendly organic semiconductors for sustainable organic electronics. J. Mater. Chem. C, 2(36), 7621-7631. https://doi.org/10.1039/c4tc00550c
Lee, J., Kim, J., Song, J. E., Song, W. S., Kim, E. J., Kim, Y. G., Jeong, H. J., Kim, H. R., Choi, K. Y., & Kim, B. G. (2021). Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nature chemical biology, 17(1), 104–112. https://doi.org/10.1038/s41589-020-00684-4
Namgung, S., Park, H. A., Kim, J., Lee, P.-G., Kim, B.-G., Yang, Y.-H., & Choi, K.-Y. (2019). Ecofriendly one-pot biosynthesis of indigo derivative dyes using cyp102g4 and prna halogenase. Dyes and Pigments, 162, 80-88. https://doi.org/10.1016/j.dyepig.2018.10.009
Regan, H. (2020, September 29). Asian rivers are turning black. And our colorful closets are to blame. CNN. Retrieved August 3, 2021, from https://edition.cnn.com/style/article/dyeing-pollution-fashion-intl-hnk-dst-sept/index.html
