Presented by Team SZU-China 2020
Authors:
Poster design: Xiaoli Long
Wiki code: Xiaoli Long
Content: Wenxi Li, Shuqi XU, Zirui Ou, Ying Xu
2020 SZU-China iGEM Student Team members : Xiaoli Long, Xinyu Li, Wenxi Li, Lingling Liao, Yan Huang, Jielin Li, Zirui Ou, Shuqi Xu, Xinyu Liu, Ying Xu, Zening Xiao, Jiaqi Huang.
2020 SZU-China iGEM Team PI, advisors: Weizhao Chen, Ying Xu, Shanshan Xu, Jiaqi Tang, Rongsong Ling, Yiming Chen, Jianzi Li, Jiantao Mai.
Abstract
Producing every ton of jeans will pollute 200 tons of water due to the discharged 2500 toxic chemical substances, causing catastrophic effects on the environment. What's worse, in South China’s Pearl River Delta, the use of traditional methods for jeans fraying such as strong corrosive chemical reagents and sandblasting cause irreversible damage to the workers’ health. This year we designed an eco-friendly, efficient system for jeans factory. Our system includes two parts. In the first part, we produced thermostable beta-glucosidase via recombinant E.coli to synthesize Gardenia Blue, a stable, environmental friendly pigment with high dyeing efficiency. After dyeing, we used recombinant cellulase (endo-glucanase) for fraying in replacement of the stone-washing process. Besides, we designed a hardware to adjust our products to jeans manufacturing process. In brief, our automatic green system can replace manual dyeing and fraying process, while reducing pollution and protecting the health of workers.
Team:SZU-China/Poster
Poster: SZU-China
BLUEism:A novel eco-friendly scheme for jeans dyeing and washing
1.Background
Low cost blue jeans actually has a high cost attached to it.
The traditional method in making distressed blue jeans consumes prodigious amounts of hazardous chemicals and water while dyeing, bleaching and fraying, posing a horrendous threat to the environment and public health, especially the workers’. To make it worse, many jeans factories with poor sewage disposal system dump the toxic wast water directly into the river[4].
And when it comes worn-look making, which includes abrasion and bleaching. The fine dust generated by manual abrasion is notoriously blamed for leading to the works’ lethal pneumoconiosis. And the process of bleaching use the strong corrosive chemicals that is also dangerous for workers.
In general, a more sustainable schemes for jeans manufacturing is in great urgency.
The traditional method in making distressed blue jeans consumes prodigious amounts of hazardous chemicals and water while dyeing, bleaching and fraying, posing a horrendous threat to the environment and public health, especially the workers’. To make it worse, many jeans factories with poor sewage disposal system dump the toxic wast water directly into the river[4].
And when it comes worn-look making, which includes abrasion and bleaching. The fine dust generated by manual abrasion is notoriously blamed for leading to the works’ lethal pneumoconiosis. And the process of bleaching use the strong corrosive chemicals that is also dangerous for workers.
In general, a more sustainable schemes for jeans manufacturing is in great urgency.
3.Our solution
Gardenia Blue: eco-friendly bio-pigment for dyeing. Cellulase (endo-glucanase): alternative of traditional method for worn-look effect.
Aiming at the two most sticky problems in jeans processing, we proposed two corresponding solutions based on synthetic biology.
We use the blue bio-pigments, gardenia blue for dying. It is derived form the fruit of gardenia. Not only does it circumvents the use of reductant, but it is much more safe to use as an edible additive. And for worn-look effect, we use celluase as an alternative way to abrasion and chemical corrosion. Besides, we designed a hardware to adjust our products to jeans manufacturing process. And we built a backpropagation neural network and other models to help manufacturers better optimize their production.
In brief, our automatic green system can replace manual dyeing and fraying process, while reducing pollution and protecting the health of workers. And we endeavor to optimize industrial techniques for producing jeans without sin.
Aiming at the two most sticky problems in jeans processing, we proposed two corresponding solutions based on synthetic biology.
We use the blue bio-pigments, gardenia blue for dying. It is derived form the fruit of gardenia. Not only does it circumvents the use of reductant, but it is much more safe to use as an edible additive. And for worn-look effect, we use celluase as an alternative way to abrasion and chemical corrosion. Besides, we designed a hardware to adjust our products to jeans manufacturing process. And we built a backpropagation neural network and other models to help manufacturers better optimize their production.
In brief, our automatic green system can replace manual dyeing and fraying process, while reducing pollution and protecting the health of workers. And we endeavor to optimize industrial techniques for producing jeans without sin.
2.Inspiration
Documentary named "Der Preis der Blue-Jeans". Investigating local jeans washing factory.
While brainstorming, we happened to see the documentary named “Der Preis der Blue-Jeans “. This film talks about how a group of environmentalists traced back the origin and life of a pair of jeans worth 9.9 Euro, during which the inside story of jeans industry was gradually revealed. Retailers keep forcing down the price for their own interests. And the manufacturers have no choice but to use cheap labor and not eco-friendly techniques for manufacturing, so as to meet sellers’ stringent demand and struggle to survive. And in this way, people usually neglect the rights and health condition of the workers and sacrifice the environment and future for short term profits.
Fig. 1 Machine covered with thick dirty dust (photographed by SZU-China)
After watching the documentary, we set out to interview a jeans washing factory in Zhong Shan for first-hand background research. The on-the-spot investigation did give us a shock, with the thick blue dust, pungent smell and nerve-racking noise from machine. And is heartbreaking seeing workers working endlessly like emotionless machines in that lethal condition.
Fig. 2 A worker abrading the jeans (photographed by SZU-China)
Current solutions for dyeing, has bio-indigo extracted from plants and exogenous bio-indigo expressed by bacteria. However, extraction has limited plant resource and production, and both of them can’t avoid the use of reductant to transform insoluble indigo into soluble leucoindigo for dying. And for the worn-look making, current sollution use laser to engrave patterns on jeans, but many of the jeans factory struggling for small margin are not supposed to afford the high price of the advanced machines.
All of these inspire us to develop a more sustainable scheme for blue jeans and world based on synthetic biology.
While brainstorming, we happened to see the documentary named “Der Preis der Blue-Jeans “. This film talks about how a group of environmentalists traced back the origin and life of a pair of jeans worth 9.9 Euro, during which the inside story of jeans industry was gradually revealed. Retailers keep forcing down the price for their own interests. And the manufacturers have no choice but to use cheap labor and not eco-friendly techniques for manufacturing, so as to meet sellers’ stringent demand and struggle to survive. And in this way, people usually neglect the rights and health condition of the workers and sacrifice the environment and future for short term profits.
Fig. 1 Machine covered with thick dirty dust (photographed by SZU-China)
After watching the documentary, we set out to interview a jeans washing factory in Zhong Shan for first-hand background research. The on-the-spot investigation did give us a shock, with the thick blue dust, pungent smell and nerve-racking noise from machine. And is heartbreaking seeing workers working endlessly like emotionless machines in that lethal condition.
Fig. 2 A worker abrading the jeans (photographed by SZU-China)
Current solutions for dyeing, has bio-indigo extracted from plants and exogenous bio-indigo expressed by bacteria. However, extraction has limited plant resource and production, and both of them can’t avoid the use of reductant to transform insoluble indigo into soluble leucoindigo for dying. And for the worn-look making, current sollution use laser to engrave patterns on jeans, but many of the jeans factory struggling for small margin are not supposed to afford the high price of the advanced machines.
All of these inspire us to develop a more sustainable scheme for blue jeans and world based on synthetic biology.
4.Design
We designed two generations of blue dye, bio-indigo and Gardenia Blue, and a eco-friendly solution for worn-out.
Aiming at reducing the demand for the use of harmful chemicals, we are wondering if we can use synthetic biology methods to produce low-cost blue pigment.
Firstly, via tryptophanase encoded by tnaA enzyme, tryptophan can be transformed into indole, the precursor substance of indigo. Secondly, under the action of the flavin monooxygenase encoded by the gene FMO, the hydroxyl group is introduced into the indole to form the intermediate hydroxyindole. Finally, when the cell is lysed, indole can spontaneously oxidize to indigo.
Therefore, recombinant tryptophanase and recombinant flavin monooxygenase are expressed by engineered E.coli to achieve the purpose of biosynthesis of indigo.
In the production of Gardenia Blue, via the catalytic action of beta-glucosidase, the extracted geniposide is hydrolyzed into genipin which combine with primary amino acids at a high temperature, generating natural blue pigments.
We chose an enzyme that has good tolerance to extreme industrial environments, a heat-resistant β-glucosidase from Thermus marinus.
We use cellulase to achieve worn-out effect. The cellulase hydrolyzes the cellulose, causing a part of the indigo dye to fall off the fabric to achieve the “worn feeling”. We chose two genes, the endoglucanase IARI-SP-2 from Bacillus subtilis and the endoglucanase CL34 from Streptomyces.
Aiming at reducing the demand for the use of harmful chemicals, we are wondering if we can use synthetic biology methods to produce low-cost blue pigment.
- Biological indigo
Firstly, via tryptophanase encoded by tnaA enzyme, tryptophan can be transformed into indole, the precursor substance of indigo. Secondly, under the action of the flavin monooxygenase encoded by the gene FMO, the hydroxyl group is introduced into the indole to form the intermediate hydroxyindole. Finally, when the cell is lysed, indole can spontaneously oxidize to indigo.
Therefore, recombinant tryptophanase and recombinant flavin monooxygenase are expressed by engineered E.coli to achieve the purpose of biosynthesis of indigo.
- Gardenia Blue
In the production of Gardenia Blue, via the catalytic action of beta-glucosidase, the extracted geniposide is hydrolyzed into genipin which combine with primary amino acids at a high temperature, generating natural blue pigments.
We chose an enzyme that has good tolerance to extreme industrial environments, a heat-resistant β-glucosidase from Thermus marinus.
- Cellulase
We use cellulase to achieve worn-out effect. The cellulase hydrolyzes the cellulose, causing a part of the indigo dye to fall off the fabric to achieve the “worn feeling”. We chose two genes, the endoglucanase IARI-SP-2 from Bacillus subtilis and the endoglucanase CL34 from Streptomyces.
5.Engineering Success
We successfully expressed the heat-resistant beta-glucosidase and produced gardenia blue in large scale, and dye the denim fabric which dyeing quality coincides with standards. We also successfully expressed the endo-glucanase for adding distressed detailing on denim fabric.
First generation of blue dye: Bio-indigo
Successful expression of biological indigo
During the experiment, we found that the production of bio-indigo was unsatisfactory with 0.0802mg/ml in average. In addition, we got the unexpected orange-red dye in the fermentation product which may be due to the production of isatin.
Second generation of blue dye: Gardenia Blue
Due to the shortcomings of bio-indigo, we found a better blue dye, Gardenia Blue, to replace bio-indigo.
Successful expression of recombined β-glucosidase and its enzyme activity in different temperature.
Successful production of Gardenia Blue with different amino acids.
We did single factor analysis to propose an optimal formula for pigment production, which is 6 ul glycine, 440 ul geniposide,50 ul enzyme solution react at 80 degrees for 150 minutes in 1ml system.
Successful expression of recombined IARI-SP-2 and recombined CL34 and their enzyme activities.
The surface morphology of the untreated and treated denim samples studied via a scanning electron micro-scope.
- Pigment
First generation of blue dye: Bio-indigo
Successful expression of biological indigo
During the experiment, we found that the production of bio-indigo was unsatisfactory with 0.0802mg/ml in average. In addition, we got the unexpected orange-red dye in the fermentation product which may be due to the production of isatin.
Second generation of blue dye: Gardenia Blue
Due to the shortcomings of bio-indigo, we found a better blue dye, Gardenia Blue, to replace bio-indigo.
Successful expression of recombined β-glucosidase and its enzyme activity in different temperature.
Successful production of Gardenia Blue with different amino acids.
We did single factor analysis to propose an optimal formula for pigment production, which is 6 ul glycine, 440 ul geniposide,50 ul enzyme solution react at 80 degrees for 150 minutes in 1ml system.
- Worn-out
Successful expression of recombined IARI-SP-2 and recombined CL34 and their enzyme activities.
The surface morphology of the untreated and treated denim samples studied via a scanning electron micro-scope.
- Proof of concept
7.Hardware
Trousers to blue jeans-Automatic producer.
With the machine all you need to do is place your trousers onto the trousers clamp and it can automatically goes through transferring dyeing worn-out making washing and drying.
Besides, we designed a machine to use our product. Before use, the user needs to add the enzyme solution along with Glycine and Geniposide to the dyeing pool and the abrasion pool respectively. We will produce and sell purified enzyme solution and all these above reagents can be purchased and used multiple times.
Then, the machine will automatically run to produce pigment. After 150 minutes, the user can load the trousers on the trousers clamp, and the machine will automatically complete the dyeing and other steps. When it's all done, you can go out with your new jeans dressed!
With the machine all you need to do is place your trousers onto the trousers clamp and it can automatically goes through transferring dyeing worn-out making washing and drying.
Besides, we designed a machine to use our product. Before use, the user needs to add the enzyme solution along with Glycine and Geniposide to the dyeing pool and the abrasion pool respectively. We will produce and sell purified enzyme solution and all these above reagents can be purchased and used multiple times.
Then, the machine will automatically run to produce pigment. After 150 minutes, the user can load the trousers on the trousers clamp, and the machine will automatically complete the dyeing and other steps. When it's all done, you can go out with your new jeans dressed!
6.Model
We performed molecular docking and constructed many mathematical models applied in industrial large-scale production.
In the project, we used Pymol and Discovery studio to perform molecular docking and virtual amino acid mutations on β-glucosidase to improve its enzymatic activity. In order to predict the optimal harvest time of β-glucosidase, we established a gene expression model. At the same time, we established a BP neural network model based on the orthogonal test to predict the production of gardenia blue, and then used genetic algorithm to optimize.
3D structure of β-glucosidas shown by Pymol
Comparison of predicted and true values
In the project, we used Pymol and Discovery studio to perform molecular docking and virtual amino acid mutations on β-glucosidase to improve its enzymatic activity. In order to predict the optimal harvest time of β-glucosidase, we established a gene expression model. At the same time, we established a BP neural network model based on the orthogonal test to predict the production of gardenia blue, and then used genetic algorithm to optimize.
3D structure of β-glucosidas shown by Pymol
Comparison of predicted and true values
8.Human Practice
Problems to solutions-Production line investigation-producers-experts-government.
Considering to understand the problem, we first went to a jeans factory to see the production line and the fraying process.We also culture some experts and introduce our design to get some feedback.Then we went to a textile company and this time, we place more emphasis on how to select a best dyeing pigment and the industrial steps of dyeing.At last, we connect our solution with government laws to ensure that our product is eco-friendly.
Considering to understand the problem, we first went to a jeans factory to see the production line and the fraying process.We also culture some experts and introduce our design to get some feedback.Then we went to a textile company and this time, we place more emphasis on how to select a best dyeing pigment and the industrial steps of dyeing.At last, we connect our solution with government laws to ensure that our product is eco-friendly.
9.References & Acknowledgement
- [1] Hsu, T. M.et al., Employing a biochemical protecting group for a sustainable indigo dyeing strategy.Nat Chem Biol, 2018. 14(3):p. 256-261
- [2] Zhenxuan, W., Cloning, expression and the catalytic mechanism research of a bacterial flavin-containing monooxygenase.Shanghai Jiao Tong University, 2011.
- [3] Han, G.H., H.-J. Shin, and S.W. Kim, Optimization of bio-indigo production by recombinant E. coliharboring fmo gene.Enzyme and Microbial Technology, 2008. 42(7): p. 617-623.
- [4] Zhang Hui, Qiu Riyong, Liu Mingjie, et al. Cloning and overexpression of extremely heat-resistant β-glucosidase gene and its application in the industrial production of natural blue pigment[J].Jiangsu Agricultural Sciences,2013,41(7 ):19-22.
- [5] Song Huijun, Mei Hongwen, Yin Xuchu, et al. Dyeing process of cotton knitted fabric with gardenia blue pigment[J].Knitting Industry,2019,(4):40-43.
- [6] Luo Weiguang. Cloning, expression and enzymatic properties of cellulase genes derived from Bacillus subtilis[D]. Henan: Henan University of Science and Technology, 2015.
- [7] Qiang Wang,Jiugang Yuan,Yuanyuan Yu.Cellulase immobilization onto the reversibly soluble methacrylate copolymer for denim washing[J].Carbohydrate Polymers: Scientific and Technological Aspects of Industrially Important Polysaccharides,2013,95(2):675-680.
Here, we would like to express our sincere thanks to the individuals and organizations who have helped us in different ways this season, there won’t be Blueism without your effort!