Presented by Team HKUST 2020
Cheuk Hei TSANG¹, Coral SUN¹, Derek SHAO¹, Judy CHO¹, Macy YUEN¹, Richard TSE¹, Sarah HUNG¹, Timmy WONG¹,Vanessa LEE¹
¹iGEM Team Member, iGEM Team Advisor, iGEM Team PI
Abstract
The conventional textile industry is causing noxious pollution to the hydrosphere. Non-biodegradable synthetic fibers, such as nylon, are causing microplastic pollution. While chemical dyes discarded in factories’ sewage are mostly poisoning to aquatic lives.
In view of the issue, our project targets to replace these fibers and dye by producing recombinant hagfish slime intermediate filaments (IF) with a chromoprotein dyeing system. A novel protein purification approach is also designed as an industrial solution. The hagfish slime IF is a competitive candidate to replace the polluting man-made fiber. While being biodegradable, it possesses outstanding mechanics that outscore Kevlar. When compared to other developing alternatives, like spider dragline silk, its simpler protein architecture facilitates the production process. We also developed an eco-friendly dyeing method for our threads. By fusing non-toxic chromoproteins to the IF, they can grant a wide range of vivid colors for the fabric, making it a viable clothing material.
Team:HKUST/Poster
Poster: HKUST
Poster Template
This poster template will let you create an interactive poster! The poster is divided in two parts: the visual overview on the left and the documentation on the right. The visual overview is broken down into sections that the user can click on. When a user clicks on a poster section in the visual overview, the documentation on the right will display the text and graphics associated with this section of the poster. You can find the documentation on how to use this template, as well as an example here: https://2020.igem.org/Competition/Deliverables/Poster
GREEN TEXTILE SYSTEM: Biodegradable hagfish slime intermediate filament and non-toxic chromoprotein-based dye for fabric production
Introduction
Our project involves engineering a biodegradable textile material to reduce our reliance on synthetic polymers. We find great potential in hagfish slime intermediate filaments (IF), one of the components of hagfish slime. With its amazing mechanical properties and ability to self-assemble in seawater, it is a promising substitute for synthetic fibers. We give the IF proteins vibrant colors by fusion with chromoproteins. Through mixing subunits of different colors, we hope to create a myriad of colors and tune it precisely.
Our goals are to:
Our goals are to:
- Create novel textile materials with amazing mechanical properties & vivid colours
- Alleviate the microplastic and water pollution problems from its source
- Explore an alternative to highly polluting industrial dyes
- Keep the cost of manufacturing low
Problem
Microfiber problem：
Common synthetic fibers, including nylon, acrylic, and spandex, prevails in the current textile and apparel market. Same as plastics, they are petrochemical derivatives. They can pollute the ocean in the form of microfibers, a class of microplastic. Which accounts for more than 30% of primary microplastic in the hydrosphere. Every time clothes made with synthetic materials are put into the washing machine, fabrics rub and abrade, causing little fibril shedding off. Similar to microplastics, microfibers can concentrate pollutants, bioaccumulate within organisms, and magnify along the food chain.
Additional to washing, recent research shows that wearing the clothes only also releases microfibers to the atmosphere. These microscopic airborne fibrils can escape from the primary defense system and our circulatory system once inhaled.
Dyeing industry pollution:Most of the dye do not bind tightly to the faber matrix and will be discharged directly to secluded rivers as sewage.The coloration of the disturbs the ecosystem by preventing light from penetrating through, reducing primary productivity rate, increasing biochemical oxygen demand (BOD) and chemical oxygen demand (COD) for degrading the dyeing compound. Ultimately producing anoxic zone and killing cornered organisms. Being extremely soluble in water, these dyes could hardly be removed by conventional water treatment methods. Besides, many of the abovementioned dyes are harmful to aquatic organisms. They could be transformed into more dangerous compounds, such as cytotoxins, mutagens, and carcinogens, with the help of enzymes inside an organism.
Idea
Our goal is to produce and promote a biodegradable textile material to reduce the reliance on the current synthetic polymers. We find great potential in hagfish slime threads’ amazing mechanical properties. Therefore, we hope to manufacture it as a novel proteinaceous textile material. Additionally, we also want to fuse chromoproteins on the subunits for coloring of the whole fiber. Through mixing subunits that are of different colors, we hope to explore an alternative to the highly polluting industrial dyes.
Effectiveness and the cost of manufacturing are also within our concerns. We work on the genetic circuit to boost expression efficiency in order to meet the high demand of proteins for fabrication. Besides, to maintain a reasonable cost of production, we searched for an innovative, and less expensive approach, when compared to traditional chromatography, for protein purification.
Effectiveness and the cost of manufacturing are also within our concerns. We work on the genetic circuit to boost expression efficiency in order to meet the high demand of proteins for fabrication. Besides, to maintain a reasonable cost of production, we searched for an innovative, and less expensive approach, when compared to traditional chromatography, for protein purification.
Fundamental Research
Objectives:
Protein Structure Prediction:
Conclusion:
- To predict the molecular structure of hagfish slime intermediate filament (IF) subunits and dimer.
- To demonstrate the real-time conformational changes during draw-processing at atomic level.
- To estimate the tensile properties of our product as a versatile material.
When hagfish encounters danger, it will produce the hagfish slime containing both the mucin and protein fibres. And our project mainly focuses on the polymer protein fibres whose structural unit is the dimer molecule composed of alpha and gamma subunits. It has been reported that the mechanical strength of hagfish IF fibres can be tailored by a mechanism called draw-processing. Under the pandemic situation, we were unable to carry out real experiments. Therefore, in order to explore the effects of draw-processing on the mechanical properties of hagfish slime intermediate filaments, we employed molecular modeling to predict the 3D structures and perform in silico simulations with them.
Hypothesis:Coiled coil alpha-helix dimers, excluding the terminal domains, are not only the structural units but also the functional units of IF fibres, which means that it should possess the similar tensile properties to the higher-order structure like IF bundles themselves.
Protein Structure Prediction:
With the help of SWISS-MODEL and I-TASSER servers, we successfully predicted the 3D structure of hagfish slime intermediate filament dimer. And through the quality evaluations by ProSA and Molprobity, we confirmed that this dimer model performs well in many aspects like protein geometry, bond angle, etc.
Molecular Dynamics Simulations:
After getting the predicted structures, we used the software VMD to do the MD simulations. Firstly, we carried out the solvation, energy minimization and equilibration in water not only to mimic the wet experimental experiment but also to prepare a nice starting structure for the following simulations. Next, we performed the steered molecular dynamics (SMD) with the equilibrated dimer model, which can be regarded as an in silico simulation of draw-processing.
The animation shows the real-time conformational changes during the simulation.
By treating the dimer molecule as an ideal rigid rod, we could also plot the stress-strain curve to show the tensile properties of the fibres.
- Hagfish slime IF fibres possess the nice tensile properties as a promising textile material
- The appearance of beta sheets as a critical sign
Expression
Our target:
To maximise protein expression yield
Disadvantages of the current approach for hagfish slime intermediate filament subunits production in E. coli: Hagfish slime intermediate filament subunits will aggregate and form an inclusion body upon recombinant protein expression, where cell lysis is adopted to extract proteins. However, the accumulation of recombinant protein will aggregate and introduce metabolic burden to the cell, this leads to a decrease in protein expression efficiency.
How does signal peptide help solve this problem:
Our best construct to maximise protein expression yield:
By comparing 2 strong promoters and 2 signal peptides, and their protein expression yield. We could choose the one with the highest yield.
Disadvantages of the current approach for hagfish slime intermediate filament subunits production in E. coli: Hagfish slime intermediate filament subunits will aggregate and form an inclusion body upon recombinant protein expression, where cell lysis is adopted to extract proteins. However, the accumulation of recombinant protein will aggregate and introduce metabolic burden to the cell, this leads to a decrease in protein expression efficiency.
How does signal peptide help solve this problem:
- Avoid protein aggregation and retain protein production efficiency
- With a signal peptide, the subunits are guided to the cell membrane, and signal peptide is cleaved
- Only hagfish slime intermediate filament subunits are secreted out of the cell
Our best construct to maximise protein expression yield:
By comparing 2 strong promoters and 2 signal peptides, and their protein expression yield. We could choose the one with the highest yield.
Purification
At the first phase of the project, we primarily use His-Tags for the purification of IF subunits. To reduce cost for an industrial production of recombinant hagfish slime threads as a clothing fabric, we planned to implement the Type 3 secretion system (T3SS) purification system, developed by the 2018 UCopenhagen iGEM team.
The system utilizes the synthetic injector E. coli (SIEC) as a chassis, which is a non-pathogenic strain that could express the T3SS injectisome as a molecular syringe for the translocation of a tagged protein through the inner membrane, outer membrane and an extracellular membrane. (shown below)
After the protein is secreted via the extracellular membrane, it is stabilized in the buffer solution at the collection chamber and is ready for collection. Below shows the proposed hardware for the implementation of the system.
After the protein is secreted via the extracellular membrane, it is stabilized in the buffer solution at the collection chamber and is ready for collection. Below shows the proposed hardware for the implementation of the system.
We opt for this novel purification system because of:
The system utilizes the synthetic injector E. coli (SIEC) as a chassis, which is a non-pathogenic strain that could express the T3SS injectisome as a molecular syringe for the translocation of a tagged protein through the inner membrane, outer membrane and an extracellular membrane. (shown below)
After the protein is secreted via the extracellular membrane, it is stabilized in the buffer solution at the collection chamber and is ready for collection. Below shows the proposed hardware for the implementation of the system.
After the protein is secreted via the extracellular membrane, it is stabilized in the buffer solution at the collection chamber and is ready for collection. Below shows the proposed hardware for the implementation of the system.
We opt for this novel purification system because of:
- One-step purification process, effectively reduces purification steps, and cost;
- Our keratin subunits do not require post-translational modification permitting translocation by the injectosome;
- Moderate protein size (~66kDa) for translocation;
- Intracellular folding process is insignificant. Proper folding of proteins will be guided by urea dialysis
Dyeing
Our Focus :
3 main areas in our dyeing system
- Types of Dye
- Linkage of Dye
- Colours Variety
Our target
【produce fibres in many colours + maintaining the incredible mechanical properties in hagfish IF】
✅bring our products closer to a viable material for clothing
✅reducing toxic chemical pollution from traditional dyeing.
1.Type of Dye
We have chosen 9 chromoproteins as our preferred dye.
Why chromoproteins?
✅Encoded by 1 single gene ,easy to be expressed
✅Around 24-26 kDa
✅ vivid colours in visible light
✅Biodegradable & Non-toxic
Each CP is flanked with AscI & Acc65I for inserting in different terminals of IF subunits
2. Linkage of Dye
By focusing on covalently linking chromoprotein at different terminals in each subunits, we will understand the effect of chromoproteins on polymerization of IF and able to choose the best linkage methods
Red arrows indicate CP inserting position,Acc65I is for inserting CP at N-terminal while AscI is for inserting CP at C-terminal of IF subunits
3. Colours Variety
Our RYB subtractive colour mixing system involves covalently-linking different colours of chromoproteins to each subunit. By mixing in different ratios, we hope to create lots of coloured fibres !
An example is linking blue CP on Gamma subunit while linking yellow CP on Alpha subunits at N-terminal will results in green IF fibre. This is a more intuitive and convenient way to generate large amount of colours in fibres!
HP and IHP
Human practice - Public Talk
- How to Solve Plastic Pollution: The Conservation and Synthetic Biology Approaches
Organised with the CityU iGEM team, this talk provided the public information and data on global plastic pollution and knowledge on synthetic biology, iGEM projects particularly. Gordon So, a representative from WWF-HK, also shared the organisation's solution to alleviate pollution.
- Synthetic Biology: Current Technology and Career
- Questionnaire
- Stakeholders
We are happy to be invited by the CityU iGEM team to share knowledge and facts on microplastic pollution problems and career pathways for environmental work with the high school students in Hong Kong.
Integrated Human Practice
We distributed a questionnaire on our social media pages and among our own connections to study the general public’s viewpoints on topics related to our project. Our questionnaire was separated into two sections, the main focus of the first section was to study people’ daily consumption pattern and preferences on clothing, and awareness on global microfibre pollution problems, while the second section was to collect the public's opinion on genetically modified products as well as our project.
After analysing the results, we realised most of the respondents are aware of environmental issues caused by the fashion industry, and they are willing to adopt greener and more sustainable fashion choices. They are also concerned about the price, comfortability and durability of the clothes when making decision. These are items we have to pay attention to when synthesizing the hagfish IF. Apart from that, most people do not know much about hagfish or synthetic biology. We will formulate more education plans in the coming phase.
Dr. Douglas Fudge
We had a meeting with Dr. Fudge, professor in University of Chapman as he is an expert on hagfish and has previously carried out experiments with hagfish intermediate filaments. He advised us to be more careful when developing dyeing methods for the intermediate filaments as some dye might not work well while some might affect the polymer interactions.
Esquel Group
Esquel Group is a Hong Kong-based textile and apparel manufacturing company. In our meeting, they gave us much insight on the current development of the fashion industry in Hong Kong, and brought up issues on new fabric and dyeing. Our main takeaways was our production has to be sustainable to attract people.
HKRITA (Hong Kong Research Institute of Textiles and Apparel)
During our meeting, HKRITA shared with us their business operation and projects while we presented our project idea and direction. They commented that breathability and diverse colour choices are new trends in the fashion industry, and highly recommended us to include these features when designing our hagfish product. That is why we started developing the colour mixing model, to suit the taste of the general public.
Education
- It’s About Genetics!
- Mack and Aaron’s Lab
- Ocean Rescue
- Informative posts on HagBric-related topics
- Extended Biology notes for HKDSE students
- True Facts
- More About Genetic Engineering
Board games
A game that teaches synthetic biology by turning gameplayers into “researchers” and asking them to express 3 genes of interest to win the game.
Comic
Take a close look into Mack and Aaron’s daily lab story as committee members of Science Club, and learn from the mistakes they have made.
Set at “SEA SCHOOL”, the students formed the Ocean Rescue team to educate others about marine pollution and rescue the precious marine life.
Social Media
Hagfish and faBric, together we have HagBric. We shared information and fun facts on hagfish and the fashion industry on our Instagram and Facebook page.
Textbook
With hand-drawn graphics and carefully-selected content, we designed the notes based on the Biology Electives syllabus of HKDSE and provided extended learning with previous iGEM projects.
YouTube
A deeper exploration into hagfish and its intermediate filaments with animations and a humorous-but-not-too-funny narration.
Video collaboration project with the CityU iGEM team to explain the mystery behind genetic engineering.
Future Plans
Dry Lab
(aeBlue is linked to each alpha and gamma subunits)
By applying our fusion protein 3D structural model into molecular dynamics simulation, we will obtain the Optimal Temperature of using coloured Fibres and its Colour Fading Tendency.
Human Practice- Organise synthetic biology experience day-camp for high school students in Hong Kong (physical) and other areas (virtual)
- Hold physical (game) booths on street or at school to raise public awareness on plastic pollution and our iGEM project
- Commercialise our hagfish IF product after successful synthesis in lab
- Pitch our project to textile manufacturers and clothing companies to search for potential investors and adopters
- Look for green groups that can collaborate with us on project or future development
Acknowledgements and Sponsors
Acknowledgements
Sponsors
Dr. Douglas Fudge
Gordon So, WWF-HK
HKRITA
Esquel Group
Sponsors