The basic reason for our design of producing tyrian purple by synbio is sustainable and environmental friendly.

Tyrian purple, mainly composed by 6,6'-dibromoindigo (6BrIG), is a dye extracted from the murex shellfish which was first produced by the Phoenician city of Tyre in the Bronze Age. It then spread in popularity and was adopted by the Romans as a symbol of imperial authority and status. The glands of thousands of putrefied crushed shellfish left to bake in the sun, with the overwhelming smell from the process. The resulting liquid was used to dye cloth fibres in manipulated variations of colours ranging from pink to violet. To obtain 1.4g tyrian purple, 12,000 Mediterranean snails would be killed. Its difficulty of manufacture, striking purple to red colour range, and resistance to fading made clothing dyed using Tyrian purple highly desirable and expensive.

For chemical synthesis, there are still no productive methods known for industrialization. This challenge often comes from the difficulty of introducing the two bromine atoms in 6BrIG.

First, most methods use bromine gas or hydrogen bromide for bromination, but they suffer from lack of regiospecificity, low product yield and environmentally toxic manufacturing processes. Second, when synthesis is started from brominated precursors, the high cost of these precursors becomes an obstacle for large-scale synthesis.

Therefore, the bromination process is still too inefficient and uneconomical to be feasible on an industrial scale for either biological or chemical synthesis of 6BrIG.

As a green and convenient production is preferred, syntheitc biology could be a better choice. Lee et al reported the first production strategy of Tyrian purple in E.coli using an accessible substrate tryptophan (Trp). Lee used three enzymes Trp 6-halogenase (Trp 6-Hal), tryptophanase (TnaA) and monooxygenase (MO) to finish this multiple-stepproduction.

After the background research of tyrian purple, we decided to produce tyrian purple by environmental friendly synbio synthesis. Synbio synthesis is a sustainable and highly efficient enzyme-linked reaction with non-polluting and accessible substrate tryptophan (Trp).

(2)Public survey

Besides, we did a public survey which is designed to investigate how people of different ages describe colors in their daily life, how they choose dyes, whether from natural extraction or artificial synthesis, when shopping, and how much they pay attention to the environmental pollution in their daily life.

Questions:

This questionnaire is designed to investigate how people of different ages describe colors in their daily life, how they choose dyes, whether from natural extraction or artificial synthesis, when shopping, and how much they pay attention to the environmental pollution in their daily life.

We designed both Chinese and English versions of the questionnaire. In the Chinese version, we collected the results of different age groups by spreading the QR code widely in the moments of Wechat. We put the English version of the questionnaire on Slack and collected the results from teams around the world. Here are the results:

As can be seen from the picture above, Chinese people prefer to use poetic eastern names to describe colors. However, according to the answers of the English version of the questionnaire, for foreigners, most of them only understand the meaning of Oriental names occasionally, and some of them even do not understand the meaning of Oriental names at all.

When shopping, most people don't care whether dyes are natural or not, but when it comes to choosing between natural and chemical dyes, all prefer natural dyes.

The results also show that the vast majority of people are concerned about the environmental impact of the way dyes are produced, and they will not choose the dyes which production will pollute the environment. As for how to solve the problem of pollution, more than 60 percent of people believe that "A new biological dyes derived from natural dyes should be developed to solve the pollution problem from the source". And that's one of the things about our project.

In addition, it is very important for the factory to improve the efficiency of the dye production, and our project will greatly improve the production and efficiency of Tyrian purple, which is beneficial to both the factory and the consumers.

In general, most people do not care whether dyes for daily products are natural and harmless, and they are not aware of the harm that chemical dyes can cause to the human body. Through our project, we hope that people will pay more attention to the safety of dyes when shopping. At the same time, we are trying to use synthetic biology methods to make it easier and safer to produce dyes that are harmless to humans.

Since the clothes are dyed with bio-produced tyrian purple in factories and sold directly to customers, we decided to take a survey about how will customers accept these synbio commercial products in real life.

In order to investigate the public's acceptance of synthetic biology products, as well as the current situation and dilemma of the development of the synthetic biology industry, we chose synthetic biology product artificial meat as the entry point to investigate people's attitudes towards such emerging products. We investigated the discussion data on artificial meat-related topics on the Internet and found that the public's attitude towards artificial meat products showed a trend of positive and negative polarization. Therefore, we further selected the survey group with certain relevant knowledge reserves and are the main consumers of artificial meat products - college students, to investigate their acceptance of artificial meat and its influencing factors, hoping to provide ideas for the follow-up research and development of artificial meat products, and at the same time provide reference for the further development and improvement of the synthetic biology industry. Our survey format includes questionnaire surveys, offline interviews, online data collection, and related literature review. Our research report passed the university-level selection of SUSTech and participated in the Guangdong Social Management Research Competition sponsored by the Guangdong Social Management Research Association. According to the survey results, it can be seen that although the discipline of synthetic biology has developed rapidly, the productization process is relatively slow, especially due to the impact of the epidemic, many investors consider the research and development cycle and cost, and have more concerns about synthetic biology products. The industrialization of synthetic biology still needs to be vigorously publicized and promoted.

During IHP with a natural dyeing master, Ronghua Huang, we were inspired to focus on the promotion of traditional Chinese culture through producing typical Chinese colors by synbio processes. More important, we found that these typical Chinese colors have beautiful names in Chinese but cannot be translated elegantly and well to English.

Thus, we finally had a good idea of promoting these Chinese color names in iGEM’s global platform. We hope that more global residents can understand and enjoy the charm of traditional Chinese culture.

Names of typical Chinese colors usually have a long history, which keep a lot of ancient Chinese styles. One of the biggest characteristics is that we sometimes do not describe a color by its real objects. We use names to describe the atmosphere and mood when we firstly saw these colors. Here are some examples of how we tried to describe the atmosphere and mood of these Chinese colors in English.

1.Mushan Purple：At dawn, the sky is white in the east; The sunset mist shrouded the distant mountains in purple. The distant mountain is purple. Its color seems to be in the twilight. There is a faint purple lingering in the mountains. It is soft and elegant, far away, beautiful and poetic.

2.Tianshui Bi: The imperial wife of Li Yu, the late leader of the Southern Tang Dynasty, once put the silk in the open air for the night when she was dyeing green. Because the silk was stained with dew, it changed and turned out to be very bright green. Later, people used this method to dye it and called it "Tianshui Bi", which was a kind of blue in ancient times, similar to modern light green and called jade green. We have no chance to see the color of Tianshui Bi in those years. According to the literature, this green is not dyed generally, but dyed light green. It becomes more green after being moistened by dew

3.Bamboo Moon: a moonlight scene in the bamboo forest, which is one of the traditional Chinese color blue series. It refers to the moonlight scene in the bamboo forest as a color word; It carries the noble spirit of the literati and is extremely cold and lonely. Bamboo moonlight is a feeling, so there are deep and light. A little green in blue, maybe a little purple grey. It feels different and presents different colors.

4. Red embers: charred charcoal or lake surface under the afterglow of sunset. The embers turn to ashes. Grey is a combination of black and white. If the embers are red, they are not gray, but some of them are dark red and yellow. The former is a kind of pessimistic and sad mood. When the building is about to collapse, the embers will turn to ashes; The latter is a positive and optimistic attitude. It is a kind of reincarnation that the sunset goes today and tomorrow returns. It can be described with such an elegant name as ember red, just like wild grass blowing and growing again. "The grass on the field is separated from the grass on the plain, and in one year, withers and thrives once each. Wild fires can't burn out, and the spring wind blows and grows again". There is hope in the embers. So this is the red of hope

5. Rice Yellow: Whether in the south or north, every autumn, you will see the golden patch, which is the hope. If we say that those who are often spoken of as having status yellow can only appreciate and look up. rice yellow is a true color closely related to everyone. Whether people remember its color or not, it blooms by itself every year when there is a bumper harvest

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6. Pale green: It is a deep and dark green. The first frost wood leaves are pale green, and the green has turned yellow, showing a kind of old and gloomy color.

7. Jiqing: a concise dark blue, as the color of the sky after rain. The color comes from porcelain, which is like deep sea, clean without texture, deep tone, even shade, rich and beautiful, and is an excellent representative of ancient glazed porcelain.

8. Iris blue: the color of blue irises. April is the season when irises bloom. Iris is like blue butterflies conveying the message of spring

9. Iceberg blue: The blue color appears after adding a lot of gray and white, which looks like a cool glacier in nature. This color is similar to the moon white. Wearing it in summer is more pure than white, and more refreshing than dark blue.

Pigment is a common product in people's life nowadays. It is widely used in food pigment, medical pharmaceutical, dyeing of textile industry, ballpoint pen and fluorescent ink, ink pen, cosmetics such as highlighter pen, candle, paper, pencil, pen box, pen bag and other aspects. Pigment can be divided into two major categories of natural pigment and synthetic pigments, although the natural pigment is harmless to human body, the advantages of high safety and color when using more natural, but due to the low natural pigments have a content, poor tinting strength, high cost and poor stability characteristics, is now more and more replaced by a synthetic pigment. Synthetic pigments can be divided into chemosynthetic pigments and biosynthetic pigments.

Our bacteria will be engineered to make E. coli that synthesizes Tyrian purple. We will use two E. coli as two reaction systems. In the first E. coli, we will first add bromo-group to trvptophan and knock out the tnaA gene of E. coli to inhibit other side reactions. In the second E. coli we will synthesize Tyrian purpleusing an enzyme linked reaction that is inherent in the bacteria. At the same time in order to ensure that the production time of color is what we want, we will use BGL/UGT reversible reaction to protect the pre-product, Br-indican before the generation of Tyrian purple in the last step. Tyrian purple is a very rare and therefore very expensive pigment, and this method can greatly increase the yield and thus reduce the cost of the pigment compared with extraction from mollusk. Pigments produced by the biosynthetic pathway can be widely used in people's daily life because of their convenience, high efficiency and low cost, such as dyeing textile clothes, painting and as color indicators.

1.The applications of synthetic pigment

(1) The application of synthetic pigment in food

Food pigment is a kind of pigment. It is a kind of food additive that can be eaten appropriately and can change the original color of food to a certain extent. Food pigments can be divided into natural pigments and synthetic pigments, which are usually made from chemicals containing naphthalene extracted from petroleum. The end product is called azo dyes. Synthetic pigments are cheaper, brighter and longer-lasting, so food manufacturers prefer them to natural ones (such as sugar beet extract), but most of them are toxic. Along with the development of the society, people gradually realized the synthetic pigment, the harm of human body, according to research shows that although the synthetic pigment may not directly carcinogens, but gut bacteria synthetic pigment can be decomposed into known cancer-causing molecules, and in view of the synthetic pigment the foreign substance, the body may be severe inflammatory immune response, which may cause cancer, Therefore, it is necessary to produce natural pigments that are easy to use and less dangerous. Natural pigment can be extracted from the animals and plants directly, but direct extraction is expensive, also can in the biosynthesis of microbe, microorganisms can not only produce pigment, such as red kojic rice, purple bacteria etc, also can produce pigment in plants and animals, such as beta carotene, astaxanthin, and using the microbial production of pigment, Not affected by season and weather, high yield, simple extraction process. Although the use of microorganisms to synthesize pigments has great potential and the future is promising, most of the research on pigments from microorganisms is still in the laboratory stage, and there is still a long way to go before mass production and purification. There are some experiment and laboratory successfully completed the synthetic colorants and stain in the laboratory, it has important significance in People's Daily life, because most of the commercial production of synthetic colorants are derived from oil produced by chemical methods, this can lead to unexpected health problems, especially when some dyeing agent is added to the food in time. Therefore， the researchers wanted to genetically engineered biosynthesis of color to a certain extent, eliminate the potential safety hazard chemical supplies, and the success of this experiment can largely reduce the emissions of pollutants in the process of stain, but since each produce the color of the concentration is low, want to on a large scale is applied to daily life will be very high. Common microbial pigments include carotenoids, melanins, quinones, polyketones, and indigo. ① Carotenoids are highly unsaturated compounds, insoluble in water but soluble in organic solvents, containing a series of conjugated double bonds and methyl branched chains. The color of the pigment varies with the number of conjugated double bonds. The more conjugated double bonds you have, the farther it moves to the red. At present, the main producers of carotenoids are filamentous fungi, yeast, bacteria and algae. ② Melanin is a kind of high molecular weight biological pigment, color from dark brown to black, formed by a series of chemical reactions of tyrosine, widely exists in animals, plants and microorganisms. It is usually formed by oxidative polymerization of phenolic compounds. Melanin produced by microorganisms is mainly divided into wall (membrane) combined with melanin and extracellular pigment, insoluble in acidic solution, insoluble in common organic solvents, soluble in alkaline solution, slightly soluble in water. It is often used in agriculture, cosmetics and medicine. Melanin production microorganisms also have a lot of common: bacteria, nitrogen-fixing bacteria, mold, actinomycetes and fungi. ③ Anthraquinone pigments are the largest group of quinone pigments, with about 700 compounds, which have been widely used in food and medicine. In terms of drugs, it can be used as anticancer, antibacterial and diuretic. Therefore, anthraquinone pigments have broad application prospects in food pigments. Quinone compounds are widely found in the microbial community, especially filamentous fungi. The anthraquinone pigment that has been used as a food-grade pigment is produced by penicillium oxalate, which was first discovered and named by a Czech company. (4) Indigo as a food colorant is also widely used in the food industry. However, due to the scarcity of natural indigo raw materials and the high demand for indigo in the world, the vast majority of the current circulation of indigo in the market is chemical synthesis of indigo, only a very small part of microbial synthesis of indigo and the price is very high. Chemically synthesized indigo is not allowed to be added to food products, but there are still many merchants who add it for profit, regardless of food safety. Nowadays, the safety of chemical synthetic pigment has been widely concerned in the world. How to get a large amount of natural indigo at low cost has become a hot topic for scholars at home and abroad. At the beginning of the 20th century, some scholars discovered that microorganisms could transform indole into indigo. After that, people focused on the production of indigo by microorganisms, and made a lot of research and achieved certain results. Is the cutting edge of technology is a kind of bacteria flavin dependent single oxygenase and phosphorous acid dehydrogenase expressed as fusion proteins, the applications of this kind of fusion protein to bio-catalysis, this unique catalyst abundantly expressed in escherichia coli, by the oxidation of indole and indole derivatives obtained the indigo and series of indigo blue pigment. However, this method can not be applied to large-scale industrial production because of its high requirements for instruments and equipment. At present, microbial extraction of indigo is still a hot issue, and the prospect of its industrial production is also very expected. However, it is undeniable that compared with the chemical synthesis of indigo, microbial extraction still has many shortcomings. How to make the microbial extraction method of indigo more convenient, more economical and more efficient is the direction of scholars' future efforts.

(2) The application of synthetic pigment in medicine and pharmacy

Some pigments directly inhibit the growth of bacteria, reduce the occurrence of cancer, and inhibit inflammation, while some pigments secondary metabolites of bacteria can play such a role. Anthocyanins, for example, have positive effects on health, reducing cancer risk, reducing inflammation and modulating immune responses. Pyrolysis propyl toluene is a kind of material produced by Streptomyces and Mr Charest species, have stronger immune inhibitory activity, can be used with antibiotics and anti-malaria treatment, in addition, for lung cancer, colon cancer, kidney cancer, ovarian cancer, brain cancer, melanoma, and standard 60 leukemia cell lines, are poisonous to the cells showed some potential. Purple pigment has antigenic, anti-cancer, anti-virus, antibacterial and antioxidant activities. In addition, both violet and rhodopsin have the potential to be used therapeutically in combination with chemotherapeutic drugs.

（3）The application of synthetic pigment in textile industry

Since the 19th century，the synthetic dyes is flourish, synthetic dyes and the dyeing precursor are widely used in textile printing and dyeing industry, the production process and dyeing effluent discharge of waste has caused serious environmental pollution and energy consumption problems such as (1) and part synthetic dyes have a fixed carcinogenicity and allergenic (2) in recent years, environmental protection and safety consciousness gradually improve, people natural pigment as well Environmental compatibility and biodegradability are being reemphasized. Natural pigments exist widely in many kinds of organisms, plants, animals and some microorganisms are the main sources. At present, plant pigments are widely used in the textile industry, such as tea, curcumin can be used for the dyeing of cellulose fiber and protein fiber, sorghum red pigment, mulberry and so on can be used for the dyeing of protein fiber, madder pigment, lac pigment can be used for the dyeing of synthetic fiber and protein fiber. However, the production of plant natural pigments has a certain growth cycle, and the stability of yield is greatly affected by region and season, which limits the further industrial development. Compared with plant pigments, pigments produced by microbial fermentation have a short growth cycle and good compatibility with seasons and climate, which can produce a large number of pigments of different colors and facilitate large-scale production. It has been found that the pigments produced by many bacteria and fungi have affinity for some natural fibers and synthetic fibers, and can be used for textile dyeing, such as poly-ketone pigments produced by Monascus fungi, Female quinone pigments produced by Fusarium oxysporum, and lead derivatives produced by purple bacillus. Most of the pigments show unique physiological activities, such as anti-oxidation and anti-inflammation, among which, some microbial pigments with flavonoid, phenothione and Anthracene structures have certain antibacterial properties. Dyed textiles have been proved to have certain antibacterial effects, which can be applied to the development of high-value-added textiles. As a natural, green and safe pigment resource, microbial pigment has great research value. However, most of the pigments are fat soluble and light stability is poor, which brings some difficulties to the textile printing and dyeing process. In addition, the fabric dyed with microbial pigments has some problems such as poor color fastness to the sun, which leads to challenges in the deep research and application of microbial pigments as a natural resource. The color most commonly used today as a dye in the textile industry is indigo. Chemical production of indigo requires the use of toxic chemicals, such as formaldehyde and hydrogen cyanide, and the synthetic indigo is insoluble in water, so the right chemicals are needed to make indigo useful for dyeing clothing. One of those chemicals is sodium disulfite, which breaks down into sulfites and sulfites and corrodes equipment and pipes in dye plants and wastewater treatment plants. Like plants, bacteria produce compounds called indoles, which are insoluble in water and cannot be used as dyes. By adding a sugar molecule, the indole is converted into a precursor of indigo. Glucoside can be directly converted into indigo on cloth if enzyme is added during dyeing. The basic idea of the experiment is to oxidize the indole group first to produce the highly active indole precursor. Instead of further oxidation to indigo, the hydroxyl group is glycosylated, protecting the active functional group and producing the colorless molecule indican. Sugar acts as a protective biochemical group here, stabilizing the active indoles in the reduced state until treated with B-glucosidase. In special cases, an enzyme called β -glucosidase in the plant can remove the protective group of indoxine and change it back to indoxol, which in turn spontaneously oxidizes to form indigo through the indigo intermediate.

2. The safety of synthetic artificial pigment

The pigments used in medicine, medicine and food can enter the human body through eating, so they are all classified as food pigments. Medical experts found that children intake of a small amount of food pigment, although not immediately cause adverse reactions, but it is easy to accumulate in the body, resulting in chronic poisoning, causing abdominal pain, abdominal distention, dyspepsia and emaciation. Excessive pigment can also consume the detoxification material in the human body, interfere with the normal metabolic reaction in the body, thus hindering the development of children's body and intelligence. In addition, abroad has expert to still discover, children edible pigment overmuch, not only the impulse that hampers nervous system is conducted, cause children hyperactivity, and these material still is the potential inducement of cancer. Researchers in the United States, Britain and other countries have also found that many synthetic pigments have harmful effects on the human body, which may lead to reduced fertility and abnormal births. Some pigments may be converted into carcinogens in the human body. Especially azo compound synthetic pigment carcinogenic effect is more obvious. Azo compounds in the body decomposition, can form aromatic amine compounds, aromatic amine compounds in the body after metabolic activities and target cells may cause cancer. In addition, in addition to being toxic in themselves or their metabolites, many synthetic food colorings can be mixed with tablets and lead during production. Cream yellow, used in the past to color margarine, has been proven to cause liver cancer in humans and animals, while other synthetic pigments such as orange yellow can cause subcutaneous sarcomas, liver cancer, colon cancer and malignant lymphatic cancer. Synthetic pigments exist in the form of soluble powders, prefabricated solutions, soluble particles, pastes, colloids, or solid strips, providing a wide range of color space and can be mixed together to produce many mixed colors and forms, the most common form being soluble powders. In terms of safety, synthetic pigments will not harm health as long as they are used within the scope and standard permitted by the state. However, the current problem is that the behavior of adding pigments to food is too common. Even if the content of pigments in a certain food is qualified, consumers may consume a large number of foods containing the same pigments in their daily life, which may still lead to excessive intake of synthetic pigments, resulting in behavioral abnormalities and learning disabilities. In order to ensure the safety and health of the people, the state has made strict regulations on the varieties, scope and amount of artificial synthetic pigments allowed to be used in food. At present, the common artificial synthetic pigments approved for food include carmine red, amaranth red, lemon yellow, sunset yellow and bright blue. "Health Standards for the Use of Food Additives" in our country clearly stipulates the scope and amount of food coloring in food, for example: sunset yellow, lemon yellow maximum use is 0.1g /kg, carmine only allowed in red sausage casings, soy milk drinks, shrimp chips, candy coating and ice cream, the maximum use is 0.025 G /kg, soft drinks (such as carbonated drinks), preserves, pastry, candy, jelly, etc., can use the prescribed food coloring; Artificial pigments are not allowed in milk, pure water, meat products (such as dried meat, preserved meat, pork floss), fried goods (such as melon seeds, pine nuts), and any artificial pigments are prohibited in infant products.

3. Safety test of synthetic artificial pigment

In order to strengthen the management of edible synthetic pigment in recent years, the state attaches great importance to the safety of edible synthetic pigment, the toxicity and security of the food pigment synthesis research, evaluation, supervision and already become an important work, relevant regulatory authorities have its use range limit, all kinds of pretreatment and detection method also appeared, and gradually perfect.

① High performance liquid chromatography
Synthetic colorants in food were extracted by polyamide adsorption method or liquid-liquid distribution method, made into aqueous solution, injected into high performance liquid chromatography, separated by reverse phase chromatography, qualitative according to retention time and quantitative comparison with peak area.

② Thin layer chromatography
The principle of TLC is based on the water-soluble acid synthetic colorant is adsorbed by polyamide under acidic conditions, and desorption under alkaline conditions, and then separated by paper chromatography or TLC, qualitative and quantitative comparison with the standard. This method has the characteristic of simple operation.

③ oscillopolarography
Synthetic colorants in food produce sensitive polarographic waves at a mercury drop electrode in a specific buffer solution, the wave height is proportional to the colorant concentration. When there is one or two more colorants in the food which do not affect the determination, it can be used for qualitative and quantitative analysis. The disadvantage is that there are more colorants or interference, will not be accurate determination.

④ Other methods
Other methods for detecting synthetic pigments include microcolumn, derivative voltammetry, capillary electrophoresis, electrostatic ion chromatography, and spectral scanning.