What is fashion?

Iris Apfel, the "First Lady of fabric", whose fashion is a clever combination of clothes and accessories by color, texture and pattern, once said: "Fashion is more than trends.” So it can be said that fashion is about personality, about your personal attitude.

But fashion shouldn't just be about personality, it should also be about the environment. It is reported that the textile industry produces about 70 billion tons of dye-containing wastewater each year, and the resulting waste can be at least 50%. A large amount of dye-containing wastewater is directly discharged without proper treatment, or even directly used for farmland irrigation.[1, 2] Synthetic dyes discharged into the aquatic environment will cause water discoloration, prevent visible light from penetrating the water, thus affecting the photosynthesis of aquatic plant groups, further disturbing the local food chain, and greatly damaging the entire aquatic ecosystem[3]. Moreover, textile waste usually has biological toxicity, which may come from the dye molecules themselves, such as diazo compounds[4], or from metallic parts, such as chromium in acid dyes or copper in direct dyes[5]. These poisons can cause serious health problems in humans through bioenrichment and amplification.

During the 28th Conference of the Parties to the United Nations Framework Convention on Climate Change (COP28) in 2023, a sustainable fashion show was held at the in Expo City, with the intention of awakening the public to the concept of sustainable fashion. Therefore, how to develop personalized fashion while reflecting the concept of sustainable development has become an important focus of today's fashion.

To reduce production waste and pollution, we aim to use violacein as a dye and implement in situ natural pigment production and dyeing on fabrics through E. coli. This approach promotes environmentally friendly and safe fashion items. Additionally, we plan to enhance bacterial pigment production via RNA scaffolding, protein rational design, and metabolic pathway optimization. To further personalize the experience, our app will allow users to choose their preferred dyeing patterns.

Violacein, slightly soluble in water, good stability under normal environment; It has affinity for some natural fibers and synthetic fibers, and can be used for textile dyeing, and the dyed textiles have certain antibacterial effect. In addition, violacein is a naturally occurring compound that does not negatively affect the ecosystem and is widely used in agriculture, food processing and other fields.

The whole biosynthetic pathway of violacein involves a total of 6 steps, consisting of 5 enzymatic reactions and 1 non-enzymatic oxidative decarboxylation reaction, among which the reaction catalyzed by VioE is the rate-limiting step.

With the guidance of the central rule, from micro to macro, we used directed evolution to modify the promoter, optimize the codon to adapt to the codon tendency of Escherichia coli K12, semi-rational protein design to improve the catalytic activity and stability of Escherichia coli K12, and modify the metabolic network model of Escherichia coli K12 to adapt to the production needs of violacein. Through this systematic transformation, we succeeded in greatly increasing the production of violacein. In addition, we use a protein skeleton based on RNA phase separation to bind enzymes tightly, thus enhancing the efficiency of substrate transport between different enzymes.

We design two systems for our dyeing: customized system and mystery system.

The customization system can be split into three modules—archiving, memory and reading—and basically two plasmids are included: one with several different loxP-recorded RNA expression units, the other with VioC under control of those RNAs via riboswitch blocking RBS sequence.

For archiving module, we use modified HYER[6] without hydrolase activity to block loxP sites of archives. When an archive needs to be opened, we add the corresponding RNA to release HYER to open the target through chain replacement principle, so that the loxp sites can be edited by Cre.

For memory module, blue light is given to some of the pixels according to light and shade of the customized image. N- and C- terminal of Cre will recombine under blue light[7], acquiring recombinase activity. Whether the terminator between loxP sites is cut off decides whether to express the downstream control RNA.

For reading module, the sign with which one archive differs from other ones is a hairpin. It hides the sequence that open the riboswitch of VioC, and can be opened by adding the right RNA, exposing the foresaid sequence and leading to normal expression of the enzyme.

Using this system, we plan to achieve customized service with pixel pattern memory based on Cre enzyme in response of blue light and bio-disk mainly depending on RNA.

The Mystery system is divided into random and gradient modules, each composed of controller cells and producer cells. Controller cells utilize C4-HSL and salicylic acid as signaling molecules to guide the pigment production of VioC and VioE in the producer cells.

In the random module, we employ the Cre-LoxP system to achieve randomness in hue and tone presentation. Symmetric LoxP sites facilitate diverse recombination outcomes. [8]Each LoxP site is flanked by promoters of varying strengths, controlling the expression of signaling molecules. Under blue light, Cre recombinase, activated by the light, randomly excises or inverts the promoters within the LoxP sites, generating diverse combinations of signaling molecule expressions to control different hues of pigment production.

In the gradient module, a red light-responsive promoter is used to achieve finer variations in tone. Upon sensing varying intensities of red light, the controller cells express signaling molecules at corresponding levels, directing the production of pigments of different intensities.

To design more structured and distinct patterns, we use a green light-responsive promoter to control the pigment production of VioA, VioB, and VioD, thereby creating clearer outlines.

With this system, we aim to create the “Fantasy series” based on the Cre-LoxP system and optogenetics.

[1] Efects of textile dyes on health and the environment and bioremediation potential of living organisms[J]. Biotechnol Res, 2019, 3:275–290.

[2] Kant, R., 2012. Textile dyeing industry an environmental hazard[J]. Nat Sci. 4 (1), 22–26.

[3] Krishna Moorthy, A. et al. Acute toxicity of textile dye methylene blue on growth and metabolism of selected freshwater microalgae[J]. Environ Toxicol. Phar. 2021, 82:103552.

[4] Novotny, C., Dias, N., Kapanen, A., Malachova, K., Vandrovcova, M., Itavaara, M., Lima, N. Comparative use of bacterial, algal and protozoan tests to study toxicity ofazo- and anthraquinone dyes[J]. Chemosphere,2006, 63 (9), 1436–1442.

[5] Ozturk E, Yetis U, Dilek FB, Demirer GN. A chemical substitution study for a wet processing textile mill in Turkey[J]. J Clean Prod, 2009, 17:239–247.

[6] Liu ZX, Zhang S, Zhu HZ, Chen ZH, Yang Y, Li LQ, Lei Y, Liu Y, Li DY, Sun A, Li CP, Tan SQ, Wang GL, Shen JY, Jin S, Gao C, Liu JG. Hydrolytic endonucleolytic ribozyme (HYER) is programmable for sequence-specific DNA cleavage. Science. 2024 Feb 2;383(6682):eadh4859. doi: 10.1126/science.adh4859. Epub 2024 Feb 2. PMID: 38301022.

[7] Sheets MB, Wong WW, Dunlop MJ. Light-Inducible Recombinases for Bacterial Optogenetics. ACS Synth Biol. 2020 Feb 21;9(2):227-235. doi: 10.1021/acssynbio.9b00395. Epub 2020 Jan 21. Erratum in: ACS Synth Biol. 2020 Oct 16;9(10):2857-2859. PMID: 31961670; PMCID: PMC7393974.

[8] Cautereels, C., Smets, J., De Saeger, J. et al. Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts. Nat Commun 15, 1113 (2024). https://doi.org/10.1038/s41467-024-44996-8