Reflections
Our values and why we chose the Colorfold project
The choice of which project to pursue was shaped by several factors. Along with requiring a focus on synthetic biology, there was a desire in the team for it to feature both bioinformatics and laboratory work since participants were hoping to learn different skills. Another large part of what guided us was our values and the role of the project in our society. Among many ideas, the ones that were prioritized held the potential to do good at a large scale, such as reducing environmental degradation or treating illnesses, and ideas that tried to do something new and innovative.
This focus led us to a research team at our university studying environmental samples and how they respond to the arising challenges of environmental changes. Many of their isolates from environmental samples were exhibiting strong colors and they generously offered to share their colorful bacterial strains with us. The possibility of collaborating and contributing to each other's work appealed to us and we began thinking of what could be done as a project with these strains. Our team then carried out discussions with faculty and within the team to make sure this was a project that could fall into our foremost criteria of working towards solving a real world problem. We liked the ColorFold project mainly for the potential environmental benefits, since the development of new biological dyes with varying (engineered) properties might be able to replace existing synthetic dyes, the latter contributing to the large environmental footprint of the textile industry. It also appealed to us for the possibility of furthering scientific knowledge, either through discovering new biological color molecules or through the improvement of existing molecules, since they are sometimes used as tools in synthetic biology. We also liked the prospect of our project having a broad focus on bio-dyes with multiple subparts, such as investigating the colors of the environmental isolates and also working on improving amilCP. This enabled us to shift focus into the parts of the projects that were working out and thus raising our chances of reaching interesting conclusions during the limited time.
How does our approach compare to alternative solutions?
Pigments from microorganisms are readily available in nature, but there is an issue with production in large quantities. Using microbial pigments for dyeing has been of interest for decades (Shirata A et al. 2000), and there is research on making larger yields of dyes from microbes, but there are long ways to go before this could be viable in large-scale production (Aruldass et al. 2018). Many of these approaches also focus on extracting small pigment molecules rather than proteins, which has been the main focus of the ColorFold project, which is one way we set ourselves apart.
Our approach could aid in the discovery of new chromoproteins that hold the potential to replace some of the hard/dangerous chemical dyeing products. Through bioengineering these could then also allow for making chromoproteins new traits or combining existing ones, which could increase quality, sustainability, and/or efficiency. The current way to dye fabrics is to use a variety of chemicals, which are environmentally bad but cheap and easy to use in large quantities. This has become a large problem and there are efforts to degrade contaminants from water systems that originate from different steps in textile processing, including dyeing (Shindhal et al. 2020, Jorge et al. 2023). It is important to work on cleaning up water, but it is also important to find processes that lower the amount of contaminants to begin with.
Reflection on our achievements
As presented in the results pages for both the amilcp project and the environmental strains project, we were able to make progress that’s useful in the context of bio-dye production. Finding more mutations that cause color change in AmilCP highlights how it is possible to increase available colors from already known sources, and shows how bioengineering can create colors without synthesizing them through environmentally harmful processes. Working with our Chromosearch pipeline together with the lab work on the environmental strains highlights just some of the ways novel bio-dyes can be discovered. The partly characterized colors from our environmental strains 91 and 350 is a hopeful initial step towards a process to cause more bio-dyes to be available on the market.
With more time, we would have liked to show a practical use for these colors. As a whole, we are happy to have gotten the results we did, but we did not purify as many colors as we would have liked, or get to our initial goal of transforming any of the environmental colors into E.coli. We think this process, of finding novel colors from the environment and transforming them into an expression host, is quite viable but simply needs more time and work than we had planned for.
Through our research and the interview we did with senior lecturer Nils-Krister Persson from the Swedish School of Textiles at the University of Borås, we understood that the dyes used in the textile industry are a smaller part of the dyeing process than many may think. There are many other chemicals being used for things like binding, strengthening, viscosity, and finishing by adding shine or making the fabric more water-proof (Shindhal et al. 2020). These chemicals are used in larger quantities than the actual dyes and also cause environmental damage, so this may be a main focus for future studies. We are happy to have focused on specifically proteins and bioengineering since this enables for interesting work on the properties of the chromoproteins. To mention some potential angles of attack; is there a way to engineer bio-dyes so they are easier to adhere to fabric? Could we make the dyes contain properties, such as shine, that would lower the need for finishing processes in the dyeing industry? We want to highlight these issues as potential projects for future iGEM teams and startups.
Responsibility
Affected communities
With this project we wanted to take some initial steps to a future with reduced production of synthetic dyes, which are the current industry standard. By discovering and engineering biological molecules that can serve as a replacement, the main large-scale impact would be an improvement of aqueous water systems through reduced waste from e.g. textile industries, and improved health for the workers who are exposed to harmful chemicals on a regular basis (Yusuf 2019). While changing to more environmentally friendly processes would bring benefits, it might also negatively impact those who make a living from the current production and processes. There is a lot of textile dyeing taking place in India, Bangladesh, Sri Lanka, and Vietnam, where it creates many job opportunities and helps the countries’ economy (Jorge et al. 2023). Depending on the degree to which bio-dyes would replace an existing coloring agent without further need for changes in the dyeing process as a whole, compared to if it would require larger changes to the procedure, it might be that new factories are made and possibly in other locations. The textile industry and the dyeing is large and complex, so there is a lot to consider and many people involved. We have taken this in mind but this is not reason to stop working on advances in the scientific field that may improve the environment and human health.
How could our project be misused?
Misuse of the ideas we present in the ColorFold project could come from the fact that we encourage the discovery of novel bio-dyes from environmental samples. Environmental samples could contain organisms that are pathogenic to humans or other organisms, or that have the potential to cause ecological damage when introduced to a new environment. We want to clarify that in our project, the organisms had gone through a safety screening by both the research team at Uppsala University that selected them for us, and evaluators at iGEM. Since we were given plates with isolated organisms taken from the environmental samples, we never came in contact with the latter. In our Methods section for the environmental strains we clearly state that our samples were taken from a laboratory setting, not taken from the environment by us. We also state on the safety page that we carefully selected what strains to work with, and that we did consult the iGEM safety committee before beginning any lab work.
We have no reason to think that there were any safety issues with the sampling done by the research group at Uppsala University, the isolates they chose as suitable for us to work with, or the work we did with them. We believe the greater risk is how this project might encourage more environmental sampling, if they do not have access to screening and other safety precautions as extensive as we had. We must assume that anyone inspired by our efforts also will follow the rules and regulations that apply to lab work as a whole, and not do any work with environmental strains that pose a risk to anyone's safety.
Responsiveness
Both before choosing our project and during our lab work we have had conversations with people from different areas of expertise to learn more about what real-world issues exist around dyeing today. This has greatly expanded our view on our project and its possible uses, and guided us through our work. Below we present who we have contacted and what they taught us.
Anthony Forster, Professor at Uppsala University
Anthony Forster is a professor in Synthetic biology at the Department of Cell and Molecular Biology at Uppsala University, who has guided many Uppsala iGEM teams throughout the years. When discussing what project to do we asked him for input about what he thought was something that would benefit society, what was in demand, and what was feasible to do in the lab during the summer. He was often present at our weekly briefings and planning meetings, and his expertise has greatly helped and guided us throughout the project.
We first thought about focusing on only discovering new bio-dyes from bacteria, but after discussion with Forster we were advised to widen the project. He brought to our attention that one problem that exists today is the slow maturation of certain chromoproteins, creating practical issues with upscaling. One chromoprotein, amilCP, we had at the University. This made us expand on the project, focusing on working on more parts of the dyeing industry, since there are a lot more problems than just a lack of available biological colors. This introduced the amilCP part of colorfold!
Swedish iGEM conference
At the end of June, six of our team members attended the Swedish iGEM conference in Lund, hosted by the iGEM team from Lund university. We gained invaluable experience by discussing iGEM with other people and presenting all of our projects. Every team gave input and asked questions about where our projects were currently at.
How it changed our project
The conference mainly helped our team with parts of the iGEM competition not relating to how our project will affect the world. But there were discussions about the human practices aspect of our work and the input of the other participants was valuable and thus the conference is presented here under human practices. For instance, we got inspiration from what people the other teams had contacted for their human practices work and responses they had gotten, what everyone thought was most important when it comes to human practices and how other teams saw the impact of their project on the world. This was also the beginning of the board game collaboration that includes presentations of how all of our projects are considering their potential to improve a real world problem.
Visit at Testa Center, Uppsala
In June, the whole team spent an afternoon visiting Testa Center, where we got an in-depth look at what they do and how they support companies aiming to scale up biological production. Testa Center is a unique concept in Europe which operates as a collaboration between Cytiva and the Swedish government, facilitating equipment, guidance and essential resources for startups, small businesses, and university research groups to move from lab-scale experiments to larger production processes.
Testa Center works with a wide range of clients, from early-stage startups to research groups at universities. Most clients come to Testa Center when they’re ready to scale up their production, moving from bench-top experiments (like those in shaker flasks) to larger volumes. Projects typically last around five weeks, though microbial-based projects can often be completed more quickly due to shorter generation times. Clients can also bring their own materials for purification, though there is a minimum charge for at least one week of work.
What we learnt
Upscaling: A major part of the visit focused on what companies need to consider when scaling up production. One of the first decisions is choosing the right expression system. Testa Center works with widely-used systems like E. coli, CHO cells, and hamster cells. The choice of expression system can significantly impact yields and process efficiency, and Testa Center provides advice on which system is best suited to each project.
When scaling up, the choice of bioreactor is also critical. Options range from stirred tank reactors to more specialized ones like jet-loop or propeller-loop reactors. Testa Center helps clients select the right type of reactor, as well as the best operational mode, whether that’s batch, fed-batch, or continuous. They recommend taking things step-by-step, starting with smaller volumes and gradually increasing the scale to minimize risks.
One of the challenges discussed during the visit was the issue of protein toxicity and inclusion bodies during large-scale production. Inclusion bodies, where proteins misfold, can be particularly problematic and aren’t easy to fix once they form. This issue can complicate the scaling process, especially for projects where protein quality is crucial. While choosing the right expression system or bioreactor can reduce some costs, these aspects are often time-consuming and require careful planning.
Safety
Testa Center operates under strict safety protocols, particularly when it comes to biosafety level (BSL) requirements. They can accommodate projects involving BSL-2 organisms, but this adds extra safety considerations, especially at larger production scales.
To prevent contamination, all bioreactors are sterilized before use—either by autoclaving (for smaller reactors) or steam sterilization for larger systems. They also weld nutrient bottles directly to the bioreactors to ensure sterility throughout the process.
Nils-Krister Persson, Senior Lecturer at the Department of Textile Technology at the University of Borås.
We contacted the Swedish School of Textiles at the University of Borås, the only University in Sweden with education in modern textile design and production. Three of our members met senior lecturer Nils-Krister Persson in an hour- long video call. He works at the institution of textile technology and has published research in areas such as electrically conducting textiles.
Our main questions were about the current industry and how aware people are of the issues with textile dyeing. Since the Swedish School of Textiles has their own dyeing lab, we also wanted to ask how we could prototype a dye if we were to make one.
What we learnt
Early in the discussion Persson encouraged us to think about why microorganisms would produce color, thus inspiring a more holistic view on the concept of bio-dyes. We had a short discussion about functionality from an evolutionary perspective (antibiotics, sun protection, chlorophyll, etc.).
Persson tells us that 99% of textiles are dyed, so it is a relevant topic. We ask what he thinks about the general awareness about the issues with dyeing and the concept of biodyes. He says that the average consumer is probably not very aware of the issues but there is definitely an awareness in the textile community. About bio-dyes, he thinks that even the textile community may not be involved in what it is. The term is used in research and there are conferences and a lot of research currently and Persson is aware that there are efforts being made in the field to make dyes that are not fossil-based.
Persson strongly emphasized the complexity of the dyeing process, involving many other dangerous chemicals other than just the dyes. Many issues also occur due to other aspects of dyeing, such as the worker conditions. So even though he understands why it is more fun to work with the actual colors, they are only a very small part of improving the industry. This was not news to us, but we had underestimated how extensive the process was and that many other chemicals were used in larger quantities than the dyes. A result of this meeting was that we got more excited about the bioengineering part of our project, since we started thinking about possibilities to include other functions in an engineered chromoprotein to make it multi-functional. This may mean that the protein both produces a strong color, and also has adhesive properties to decrease need for adhesive chemicals, or have properties that enables removal of the color to improve recyclability of the textile. Persson was positive towards such projects and said it may be a great way to lower waste in the textile industry. He also brought to our attention that many colors used today are UV-sensitive. This means that it might be interesting to involve color-supporting groups into a dye molecule that could absorb UV-energy. We did not have time to work on engineering such functions over the summer, but we did get a stronger understanding of how engineering could solve such issues in the industry.
We asked about what would realistically be required to be able to industrialize bio-dyes found through projects such as ours. We learnt that water-solubility is a requirement for most industries, and that products need to be labeled with exact particle size to be sold. Our strains 91 and 350 did turn out to be water soluble, and even though we did not get them fully characterized, we found an approximate protein size, meaning our work with them is on the right path towards being useful for dyeing.
According to Persson, there is no real problem with potentially using a dyeing lab to test out any colors we may extract and that the regulations for dyes is not something we have to worry about for the most part. But the industry has a lot of standards, where they want certain properties to be to a certain level. This includes, for instance, how well the color stays on the fabric and how the fabrics durability changes during dyeing. Children's clothing also has even more standards.
The general issues on getting natural dyes used in large scale today does, according to Persson, have a lot to do with economical and practical issues. A big issue is that dyeing often takes place in developing countries that focuses more on building the economy. Another issue is consumer acceptance. The main reasons for this is that the non-synthetic dyes today don’t produce as strong colors as the synthetic dyes, and that they are often not as long-lasting, meaning they are less attractive to purchase. These are all issues suitable to address with projects such as ColorFold, and Persson suggested highlighting engineering potentials to reach the standards. Costs can also be lowered by using byproducts. For example, the Swedish School of Textiles at the University of Borås are currently working with color from algae that are imported from other industries. We also got the advice to contact someone from the textile industry to get input on what hurdles may exist from an economic and marketing standpoint, which led us to contact Newbody (see the section below).
Also regarding the bigger picture with getting bio-dyes into the larger market, Persson phrased it to us like there are two possible approaches. Either the aim is to make a more environmentally friendly dye that as closely as possible mimics current dyes, allowing for a simple replacement of the dyeing product while keeping the same fabrics and methods that are used today. The other approach would be more radical, where the benefit of changing to bio-dyes would be so great that new entire dyeing processes would replace old ones. We understood that the latter would take a lot of work and at the stage we currently are at with ColorFold we do not strongly align ourselves with any of these approaches since we are not at the stage of implementing our dyes in the industry.
Linnea Eklann, product developer at Newbody
We contacted Newbody, a Swedish company aimed at making clothes that school classes and sports teams can sell so that they can make money for school trips or sports cups. The clothes are described as socially and environmentally sustainable, which is why we reached out to them to discuss their views on dyeing and the market for sustainable products.
We got in contact with Linnea Eklann, product developer who studied at the Swedish School of Textiles at University of Borås and have worked in the industry for many years. We met in a video meeting where our main goals included finding out if the subject of bio-dyes were a topic of discussion in the industry, how much the clothing companies are involved in how the clothes are dyed, and what is important for sustainability in the clothing industry as a whole.
What we learnt
Eklann said that there is not really any talk about dyeing practices in the work she is in, but she did learn about it in her education. She mentions that she herself has little knowledge about what bio-dyes entail. This confirmed Persson's understanding of the subject, that it’s mostly a topic in academia with a long way to go to be discussed by industry.
Newbody, and most companies in general, does not have involvement on the level of choosing dyes, more than asking the supplier for a clothing piece in a certain color with certain properties. The main reason is because these practices occur abroad and through their suppliers´suppliers, far from the brand’s own offices. This applies to the other companies Linnea has worked for and it's her understanding that practically no brand makes decisions about such details. But in general the brand Newbody does try to go the extra step for sustainability, so if they did have the choice and there was not a huge extra expense, they would have the interest to choose the “better” dyes. It's just that it does not come up in their decision making as it is now.
Customers know extremely little and the interest is low. During the 3,5 years Linnea has worked at Newbody, one or two people have asked about what dyes are in the clothes. We asked if she thinks it is possible to get customer interest in dyes if that is marketed the same way companies now market for example slow fashion. Eklann told us that in general, customers are just interested in buying things that are “good”, without really knowing what that means. Customers want to care, but are not informed enough to make good decisions in this space.
When asked about other areas where the textile industry needs to improve to become more environmentally friendly, Eklann focused on greenwashing. Things are happening in EU regulations to prohibit greenwashing, something she thinks is great since the consumer does not have a lot of knowledge. Before it has been, as Eklann called it, “the wild west” where clothing companies could claim more or less anything they want about sustainable clothing.
She also brings up the CO2 emissions and Newbody is part of STICA, The Scandinavian Textile Initiative for Climate Action, that makes reports on CO2 emissions. At Newbody over 90% of CO2 emissions occur outside of Sweden, and this is about the branch standard. This is a problem in the countries of production, where local politics affect production practices. They may prioritize other things than eco-friendly practices, such as improving their welfare. Because of this there is not a lot that can be done from the brands perspective, at least not companies of the size of Newbody. Even if they are not a small brand, they do not have enough impact, and very very few companies do.
To summarize; the main takeaways were that for a real change to occur, we need rules that could put pressure on the chain of suppliers, something similar to the European Union's REACH regulation (rules on the registration, evaluation, authorization and restrictions of chemical substances). Eklann does not know exactly how Newbodys suppliers choose their suppliers, but it is probably affected by the local market. This is why these questions are so hard for a Swedish company to impact things like dyeing processes. But in a more positive light, it’s a very small amount of the total production costs that goes to the dyeing so it is not impossible to picture that a more expensive dyeing method could be chosen, since the increase in cost of the final product would likely not be significant. At least “normal” clothing brands (that are not in the position of Newbody who has to keep sales through school classes and such in consideration when pricing products) could do this, but Eklann assumes that this is conditional on the bio-dye not being lesser than the synthetic alternative. For us, this means that work like ColorFold where we want to find stable, strongly pigmented bio-dyes, is important if we are ever to reach that point.
References
- Aruldass CA, Dufossé L, Ahmad WA. 2018. Current perspective of yellowish-orange pigments from microorganisms- a review. Journal of Cleaner Production 180: 168-182.
- Jorge AMS, Athira KK, Alves MB, Gardas RL, Pereira JFB. 2023. Textile dyes effluents: A current scenario and the use of aqueous biphasic systems for the recovery of dyes. Journal of Water Process Engineering 55:104125.
- Shindhal T, Rakholiya P, Varjani S, Pandey A, Ngo HH, Guo W, Yong Ng H, Taherzadeh MJ. 2020. A critical review on advances in the practices and perspectives for the treatment of dye industry wastewater. Bioengineered 12: 70-87.
- Shirata A, Tsukkamoto T, Yasuj H, Hata T, Hayasaka S, Kojima A, Kato H. 2000. Isolation of Bacteria Producing Bluish-Purple Pigment and Use for Dyeing. Japan Agricultural Research Quarterly 34: 131- 140.
- Yusuf M. 2019. Synthetic Dyes: A Threat to the Environment and Water Ecosystem. Shabbir M (ed.). Textiles and Clothing : Environmental Concerns and Solutions, p. 11-26. John Wiley & Sons, Newark.