Human Practices

'Good communication is as stimulating as black coffee and just as hard to sleep after'

- Anne Morrow Lindbergh, American writer, aviator

Introduction

Using synthetic biology to tackle real-world problems is a core idea of the iGEM competition. By engaging with the public, stakeholders and especially experts, the project is continuously adjusted and refined. Integrating the insights gained from these interactions is a crucial and ongoing aspect of the project's development.

This page provides a summary of all consultations and events we carried out in terms of Human Practices. We thank all participants for their valuable input and feedback. We are very grateful for all the conversations, discussion, and meetings that helped us in refining our project.

Specification of our Project Idea

At the start of our project design phase, we needed to specify our project idea in more detail. Additionally, we aimed to get some feedback and advice on our concept and its feasibility. Therefore, we contacted local experts in different fields to get various opinions.
First, we talked with Prof. Dr. Thomas Walther who is a leading bioprocess engineer at the TU Dresden. We explained our project idea and discussed the overall concept of degrading textile waste using enzymes like cellulases and PETases. He endorsed our idea and gave us feedback on the whole process. In addition, we consulted Prof. Dr. Thorsten Mascher, head of General Microbiology in Dresden, as well as Dr. Christoph Loderer, junior group leader at Molecular Biotechnology in Dresden. We discussed the details of our expression strategy which significantly shaped our project idea.

Expert talk with Prof. Dr. Thomas Walther

One of the first expert talks we had was an expert talk with Prof. Dr. Thomas Walther who is leading the Chair of Bioprocess Engineering at the TU Dresden. His research group focuses on bioprocess engineering, systems biology and synthetic biology. Our aim was to discuss with him whether our project idea would be of value to the world and suitable for the format of the iGEM competition.

The conversation with Prof. Dr. Thomas Walther confirmed that the textile waste issue is not yet optimally solved. Although the recycling of pure polymers appears to be less challenging nowadays, there are still no established protocols for the recycling of more complex materials, such as mixed textiles consisting of different fiber types. The presence of dyes, buttons and other non-textile components further complicates the recycling of clothing. Therefore, the development of efficient recycling strategies for textile waste remains a necessity.

After discussing the scope of the problem, Prof. Dr. Thomas Walther reminded us of the limited time given within the iGEM competition. He encouraged us to focus on a specific step of the recycling process, while keeping the broader context in mind. To create value from waste, the first step is breaking it down into its monomers. With this in mind, our team has chosen to concentrate on degrading the most common textile materials, such as cotton, polyester (PET), and their blends, using cellulases and PET-degrading enzymes.

In the future our project could be further developed to optimize the textile degradation and, ultimately, to gain new valuable products from textile waste. For example, the influence of the dyes and their degradation with, e.g. laccases should be considered. Moreover, a fermentation protocol could be established to convert the resulting products glucose and ethylene glycol into high-value chemicals such as dihydroxybutyric acid. The addition of regulatory systems to control the production of enzymes required for textile decomposition and further fermentation could allow the combination of degradation and synthesis reactions in one process step, therefore resulting in a consolidated bioprocess.

After reflecting on the insights gathered during our expert talk with Prof. Dr. Walther, we developed our first draft of a concept for textile waste management. In this approach, after degrading polyester/cotton materials, the resulting terephthalic acid (TPA) can be isolated for use in polyester (PET) production. Meanwhile, the glucose and ethylene glycol (EG) obtained from the process could be utilized in fermentation reactions to produce high-value chemicals.

Expert talk with Prof. Dr. Thorsten Mascher

After we chose our project idea of textile fiber degradation, we met with Prof. Dr. Thorsten Mascher who is leading the Chair of General Microbiology at the TU Dresden which focusses on molecular biology, systems biology as well as synthetic biology. He is a specialist in microbiology, particularly regarding the model organism Bacillus subtilis (B. subtilis). Our aim was to evaluate and refine our project concept with him.

Initially, we discussed potential model organisms. Since our goal is to immobilize cellulases and PETases on Bacillus spores, he recommended to test the expression and activity of target enzymes before immobilization on B. subtilis. However, Prof. Mascher proposed that, in addition to experiments with Bacillus, we should produce the enzymes heterologous in Escherichia coli (E. coli) and test them comparatively. He explained two approaches for gene expression: 1) Cytoplasmatic production of the enzymes followed by lysis and purification. 2) Secretory expression of enzymes. For the latter, he referred to a signal peptide toolbox which was established in his research group which enables testing of multiple signal peptides and finding the best fitting one for each enzyme.

Prof. Mascher gave us valuable advice how to plan our project in more detail. Instead of testing the enzyme candidates directly immobilized on spores, we decided to test their activity beforehand and only take the functioning ones for further experiments. For this purpose, we decided on performing secretory expression in B. subtilis because it facilitates the whole process without lysis and protein purification. We read through the protocol of the signal peptide toolbox and ultimately decided to not screen for the best signal peptide for each enzyme candidate because it is extremely time-consuming. Instead, we agreed to use the native signal peptides of the candidates who have one and to only produce those without a signal peptide cytoplasmatically inside the cell. These intracellular proteins can be tested by using the crude extract after cell lysis.

However, we were unsure whether it is necessary to produce heterologous enzymes in E. coli. Thus, Prof. Mascher advised us to contact Dr. Christoph Loderer who is an expert in enzymology and discuss our project concept with him.

Expert talk with Dr. Christoph Loderer

Following Prof. Mascher´s advice, we consulted Dr. Christoph Loderer who is a junior research group leader at the Chair of Molecular Biotechnology at the TU Dresden and specialized in enzymology and nucleotide biotechnology.

We explained our expression strategy including protein secretion for all enzymes with secretory signal peptide and cytoplasmatic protein production for those who do not possess a native signal peptide. He approved our strategy using secretory expression with naturally evolved signal peptides because nature provides the best combination of signal peptide and enzyme. Thus, there is no need to screen for other signal peptides. Furthermore, when we expressed uncertainty about the necessity of using both B. subtilis and E. coli for enzyme expression, Dr. Loderer clarified that E. coli is not relevant to our project due to its distinct codon usage compared to Bacillus. As it offered no value, we decided not to pursue enzyme expression in E. coli.

He also recommended starting with qualitative plate assays to verify the enzymes' activity in our initial experiments, noting that while these provide qualitative data, quantitative measurements should be obtained through photometric assays. Specifically, he advised using a glucose assay to measure cellulase activity by determining glucose concentration. Consequently, began searching for suitable plate assay protocols, aiming to apply these for detecting background enzyme activity in Bacillus strains and selecting active enzyme candidates for spore display. Once active candidates were identified, the glucose assay would be used to obtain quantitative data. Generally, Dr. Loderer´s expertise in enzymology was crucial in refining our project idea.

He emphasized the importance of signal peptides for efficient secretion in B. subtilis and suggested focusing on the immobilization of multiple enzymes on spores to create an artificial cellulosome, which eliminates the need for purification steps of heterologous enzymes. He recommended a bottom-up approach: starting with simpler enzyme systems, such as cellobiose-to-glucose conversion, and gradually adding complexity. Additionally, he encouraged focusing on monomeric enzymes due to the complexities of oligomers and highlighted the novelty of our spore immobilization concept.

Based on his advice, we expanded our research criteria to focus on selecting monomeric enzymes with phylogenetic similarity to B. subtilis, a strong safety profile, and favorable activity levels under various temperature and pH conditions. We decided to focus our efforts on directly testing bacterial cellulase candidates in B. subtilis rather than E. coli. We planned to clone bacterial cellulases in our host organism and to test bacterial cellulase candidates directly in B. subtilis using inducible promoters. Based on activity of those candidates, we would choose enzyme, which we use for the development of an artificial cellulosome on spores. Additionally, we tried to establish the glucose assay for more precise activity measurements.

Throughout the project, we plan to meet with Dr. Loderer at least once a month to seek his advice on improving our assays and advancing our work.

Enzyme Candidates and Construct Design

To evaluate which criteria are important for enzyme selection and testing, we first met with Dr. Werner who is a leading enzymologist in Dresden. We presented our project and the results of our literature research on cellulases. Dr. Werner was very supportive of our project and provided valuable feedback on difficulties when working on cellulose degradation and enzyme immobilization.
During our literature research, we recognized that fungal enzymes are most efficient for cellulose degradation. Therefore, we consulted Prof. Dr. Christiane Liers who is specialized in fungal biotechnology. We presented our project idea using Bacillus subtilis for degrading textile fibers like cellulose and discussed the difficulties of producing fungal proteins heterologously in bacteria. She gave us valuable assessment which enabled a setup of a realistic project.
Ultimately, we met with Prof. Dr. Thorsten Mascher to finalize our project concept. We discussed all input we got from various sources so far and came up with a final project design to achieve our goal of degrading cellulose and PET using B. subtilis. His expertise in microbiology and genetics was crucial in developing our initial project idea into the final design.

Expert talk with Dr. Anett Werner

Dr.-Ing. Anett Werner is head of the Enzyme Technology research group at TU Dresden, which explores the enzyme workflow from screening and production to purification, characterization and application development. The group is particularly focused on fungal-based enzymes, including cellulases and laccases.

First, we talked about difficulties of cellulose degradation. She pointed out that cellulose is hard to degrade due to its insolubility in water, requiring dissolution for enzymes to act effectively. She recommended screening many enzymes first, then optimizing them by enzyme engineering and adjusting the optimal pH and temperature. However, we decided to only use native enzymes for our project since enzyme engineering would exceed our limited capacities. She emphasized the importance of textile pre-treatment, reinforcing our decision to form a pre-treatment group within our team.

Moreover, we focused on selecting cellulase candidates. She pointed out that cellulases of Clostridium thermocellum (Acetivibrio thermocellus) are commercially available and that Pseudomonas fluorescence is widely used. For this organism, the cellulase activity did not seem to be as high according to our literature review. However, she emphasized that enzyme activity values are difficult to compare and advised us to conduct our own tests. This also gave us a different perspective on selecting enzymes more independently of activity values in the literature. Additionally, she highlighted the potential of using Bacillus for enzyme secretion in large-scale production rather than immobilization.

Thus, we discussed the differences between free and immobilized enzymes. She explained that immobilizing enzymes can lead to reduced enzyme activity. While free enzymes are more active, they cannot be reused as easily as immobilized ones. She suggested using the standard DNS assay for enzyme activity tests and offered their optimized test system. For initial enzyme tests, she recommended selective agar plates with CMC (carboxymethyl cellulose) which are stained with congo red to identify endoglucanases enzymes with the largest halo and therefore high activity. We followed her advice and began searching for plate assay protocols and later also used the DNS assay for enzyme activity testing.

Expert talk with Prof. Dr. Christiane Liers

Prof. Dr. Christiane Liers is working at the Chair of Environmental Biotechnology of the International Institute Zittau and focuses on fungal biotechnology. Her work primarily explores the biochemical properties and industrial applications of cellulases derived from various fungal organisms.

Before the meeting, we had several questions related to the selection and application of cellulases in our project. We were particularly interested in understanding which organisms are best suited for producing effective cellulases and whether bacterial systems like Bacillus subtilis could be used to express fungal cellulases. We also sought advice on process design, particularly the pre-treatment of cotton, and the most suitable activity assays for evaluating cellulase performance.

During the meeting, Prof. Dr. Liers provided valuable insights into the challenges and potential solutions for using cellulases. She highlighted the advantages of using fungal cellulases, particularly from lignodegradation fungi, due to their higher activity and specificity compared to bacterial cellulases. She also advised on the difficulty of expressing eukaryotic cellulases in bacterial systems, underlying issues with protein aggregation and glycosylation. She pointed out, that there are multiple commercial substrates available to determine activity of cellulases. She highlighted the importance of choosing proper activity assay to determine the activity of our chosen enzymes. Additionally, we discussed the possibility of combining cellulases with PETases for sequential treatment of mixed textile waste.

Based on her input, we recognized the challenges associated with expressing fungal cellulases in bacterial systems and started to consider homologous expression of bacterial cellulases as more viable approach. Her advice on the process design and activity assays also led us to refine our experimental protocols and do more detailed research on potential assays, particularly keeping in mind the pH optimization and stability of cellulases under different conditions. To produce monomeric building blocks like glucose, she suggested using commercially available enzyme to simplify the process, though she acknowledged that this may not fully align with the iGEM competition's scope.

Expert talk with Prof. Dr. Thorsten Mascher II

Coming to the end of the project design phase, we met with our principal investigator Prof. Dr. Thorsten Mascher to get his feedback on all the input we got so far. After reviewing the literature and meeting several experts, we discussed enzymes candidates, cloning strategy and construct design.
Prof. Mascher recommended to only use enzyme candidates out of the phylum Firmicutes (Bacillota) since they provide the highest chance of functioning well in Bacillus subtilis without applying codon harmonization. Therefore, we excluded all fungal candidates as well as genes from other phyla like Actinomycetota and focused on cellulases from Bacillota species. As PETase we chose a candidate that was already codon optimized for Bacillus (Xi et al. 2021).

We defined the following criteria:
•biosafety level: S1
•phylum: Bacillota (for cellulases)
•active state: monomer
•localization: extracellular (if possible)
•pH range: ≈ 5 - 8
•temperature: ≈ 30 - 60 °C

Thus, we decided on ten enzyme candidates:

Enzyme	Gene	Species	Phylum, Class
Endoglucanases	EglS EglA CelA CelG	Bacillus subtilis Bacillus pumilus Acetivibrio thermocellus Acetivibrio thermocellus	Bacillota, Bacilli Bacillota, Bacilli Bacillota, Clostridia Bacillota, Clostridia
Exoglucanases	CelO CelS	Acetivibrio thermocellus Acetivibrio thermocellus	Bacillota, Clostridia Bacillota, Clostridia
β-Glucosidases	BglA BglB BglA	Bacillus halodurans Paenibacillus polymyxa Acetivibrio thermocellus	Bacillota, Bacilli Bacillota, Bacilli Bacillota, Clostridia
PETase	BhrPETase	Bacterium HR29	Chloroflexi

Then, we talked about construct design and cloning. Prof. Mascher informed us about BioBrick Assembly, Gibson Assembly and Golden Gate Assembly. We chose BioBrick Assembly in which all parts are flanked with standard sequences called prefix and suffix containing restriction sites for cloning. Consequently, we adjusted our part design and removed all EcoRI, XbaI, SpeI, PstI and NotI restriction sites by codon exchange. Additionally, BsaI and SapI as well as HindIII sites were removed to ensure compatibility and reusability of parts, e.g. for Golden Gate Assembly and cloning with the pET system, respectively.

Finally, we had to decide on an expression system for B. subtilis. In the literature, we found vectors with inducible promoters for overexpression of target genes. Prof. Mascher suggested to test replicative and integrative expression vectors comparatively. Therefore, we chose pBS0E-xylR-P_xylA (replicative) and pBS2E-xylR-P_xylA (integrative) with a xylose inducible promoter as vectors which were previously established in his research group.
Additionally, he recommended to use a protease deficient Bacillus strain. After literature research, we noticed that the strains WB600 and WB800N are usually used for secretory expression and finally chose the eight-extracellular-protease-deficient mutant WB800N. However, another vector will be used for our final goal to immobilize enzymes on Bacillus spores including a completely new design cycle.

Spore Surface Display

In the final phase of our project, we aimed to immobilize chosen enzyme candidates on Bacillus subtilis endospores. This technology is called spore surface display. The expertise for construct design of so-called ‘sporovectors’ was provided by one of our team members, Jenny Sauermann, who had previously worked on the project ‘SporoBeads’ at the research group of Prof. Mascher. To clarify remaining questions and to discuss the cloning strategy, we met with Elif Öztel, who worked on this project as a PhD student and is currently writing her doctoral thesis, and with Lennart Scheuffler, who is currently starting his PhD on this topic. Both provided valuable feedback for the second design phase regarding spore surface display.

Expert talks with Elif Öztel, M. Sc. & Lennart Scheuffler, M. Sc.

First, we consulted Elif Öztel who is currently finishing her PhD thesis at the Chair of General Microbiology at TU Dresden focused on spore surface display. Talking about a suitable assembly strategy, she suggested to apply classical restriction-ligation-cloning and not Gibson Assembly since this method was very error-prone in previous spore display experiments.

In addition, we wondered whether we should generate B. subtilis strains with one enzyme per strain or put all enzymes onto the spore of one strain, either by fusing all to one anchor protein or to different anchors. Elif Öztel explained that all strategies are feasible and recommended the papers on the Bacillus BioBrick Box providing vectors with varying antibiotic resistance and integration loci which could be used for subsequent integration of enzymes (Radeck et al. 2013, Popp et al. 2017). We also talked with our principal investigator Prof. Mascher who agreed on the first strategy to implement one enzyme per strain first because it enables to check the activity individually. Only when all enzyme function well individually, they can be combined into one strain.
Therefore, we decided to generate the strains individually with one enzyme each. We have chosen the vector pBS1C out of the Bacillus BioBrick Box allowing the integration into the amyE locus in the Bacillus genome (Radeck et al. 2013). The genetic design was identical for each construct, only varying the enzyme coding gene. P_cotYZ was chosen as promoter as it performed best in previous studies of Elif Öztel and Dr. Julia Bartels who was a previous PhD student working on this topic as well (unpublished data of Elif Öztel, Bartels et al. 2018). The gene encoding the enzyme was N-terminally fused to the anchor protein CotY. Elif Öztel recommended CotY since it is the outermost anchor on the crust of the spore and worked well in previous experiments (unpublished data).
Moreover, we decided to only test N-terminal fusions due to limited time capacities. We chose B0014 as bifunctional terminator which was also used in the study of Elif Öztel well (unpublished data). She informed us about the possibility of testing different linkers between the target gene and the anchor. Thus, we looked up promising linkers in the literature and decided to test flexible and rigid linkers as they might affect enzyme activity.

Since the construct design is similar for all constructs and only the enzyme encoding gene varies, Elif Öztel suggested to generate a main construct with a reporter like RFP as placeholder which is cut out and replaced with each target gene. But since our final transcriptional unit will be flanked by the BioBrick prefix and suffix, we cannot use common restriction enzymes like EcoRI, XbaI, SpeI and PstI. Moreover, we did not remove other restriction sites out of the parts which does not allow us to implement this strategy.
Additionally, we talked with Lennart Scheuffler about this idea, but he also expressed concerns regarding linkers. After cutting, there would remain some base pairs next to linker sequences, which might influence enzyme activity. Thus, he recommended to apply Overlap PCR instead and to design overhangs required for assembly. In the end, we chose this method for cloning of our ‘sporovectors’. We are very grateful for their advice which helped us to create feasible construct design.

Pre-Treatment

In our project we focus on the enzymatic reaction to degrade PET to TPA and EG as well as cellulose to glucose. To have an efficient reaction, the textile fibers must undergo a pretreatment for our enzymatic reaction to happen. Our research led us to two possible treatments: (i) alkaline pretreatment and (ii) steam explosion. (i) Alkaline Treatment, also referred to as “Mercerization” is the treatment of cotton yarns or fabrics with caustic soda solution. Alkaline treatment increases the amount of cellulose exposed on the fiber surface whereby the number of possible reaction sites rises. As it disrupts hydrogen bonds in the network structure, the surface roughness is also increased. (ii) Steam explosion is a physicochemical method using high-pressure steam to break lignocellulose structures. Both processes open the cellulose structure, making it accessible for our enzymes.

Conversations with Stakeholders

As we started our project, we recognized the importance of engaging with key stakeholders to refine our approach and ensure its relevance. Our journey into stakeholder conversations began with attending the BioAnalytica in Munich and the Bonding Fair in Dresden. These events provided us with invaluable opportunities to connect with experts, industry leaders, and potential collaborators. These experiences encouraged us to initiate more targeted conversations, where we could dive deeper into the specifics of our project. We asked questions, listened to concerns, and explored ideas that we hadn’t previously considered.

Analytica Fair in Munich

On March 20th, Matti, Aaron, Max, and Lilli attended Analytica in Munich, the world's leading trade fair for laboratory technology, analytics, and biotechnology. Thanks to the support from the Faculty of Bioprocessing in Dresden, we were able to secure tickets and spent the entire day at the fair. Our goals were to find initial sponsors, gain experience in promoting our project, and identify potential partners or interesting projects. Armed with flyers, we split into two teams and engaged with several companies. Some of these, like Promega, Merck, and Thermo Scientific, ended up supporting us throughout our journey. The contacts we made proved invaluable when it came time to order lab materials. We quickly learned the importance of pitching our idea in a compelling and concise way to keep people interested. As the day went on, we became more confident and approached every booth that piqued our interest. After eight hours, we left with an extensive contact list and were thoroughly exhausted—but the experience and benefits we gained made the trip more than worthwhile.

Bonding Fair

The Bonding Fair is an event organized by the student initiative „bonding Studierendeninitiative e.V.“. It took place from the 23.04.-25.04.24 on the campus of the TU Dresden. With the Wacker Chemie AG and BASF two important players in the chemical industry were present. Therefore, we thought it is a perfect opportunity to reach out to them and talk about the potential of our project idea in an economical context but also regarding potential funding. Matti and Max went on the fair on the 24.04.24 and had conversations with the salesmen of both companies. Although they were surprised of our request, BASF and Wacker Chemie AG as well were indeed interested in our ideas. As they do not have specifically processes with spores in operation, the end products of our degradation step were definitely used in the chemical industry, they told us. We left them the contact details of our team to be transmitted to the person in charge of project funding.

In order to understand how our project could be implemented in the real world, we needed to learn more about the existing principles of textile waste management. For that we have contacted Boer Group, one of the leading organizations in textile recycling. We received more information about how the collection, sorting and recycling of used clothing looks nowadays. Furthermore, we gained better understanding of challenges our project might face in the future.

Expert talk with Boer Group

In order to understand how our project could be implemented in the real world, we needed to learn more about the existing principles of textile waste management. For that we have contacted Boer Group, one of the leading organizations in textile recycling. We received more information about how the collection, sorting and recycling of used clothing looks nowadays. Furthermore, we understood better what challenges our project might face in the future.

To recycle textile waste, it has to be collected and sorted first. Notably, the existing collection and sorting systems are currently oriented on reuse of clothes in order to reduce the total amount of textile waste. To prolong the lifecycle of old unwanted garments with good quality, they can be given away in secondhand shops , online (Deutsche Kleiderstiftung) or in the “charity clothing shops” (so-called “Kleiderkammer” in German).

Furthermore, different kinds of textiles can be put in clothing bins from, e. g., some help organizations as Malteser Hilfsdienst e. V. or recycling companies as Dohmann Textilverwertung GmbH. Textiles collected by these organizations are sorted manually first to, again, separate good-quality pieces for their reuse. Textiles of poor quality are sorted out for recycling. Before recycling, buttons, zippers etc. have to be removed. Depending on the recycling method, textiles might have to be sorted once more. Currently there are pilot-projects that aim to automate this step. Machines are being developed which are able to separate textiles according to their composition using visual (VIS) and near-infrared (NIR) spectroscopy. However, this method has some limitations. For example, it can’t be applied to reliably distinguish black textiles composed of different materials. Moreover, to determine the precise composition of blended materials with spectroscopy methods is not a trivial task as well.

In Europe mechanical recycling of clothes, e. g., for production of cleaning rags or insulation materials is widely applied. A small percentage of textile waste is recycled fiber-to-fiber since clothes production does not mainly happen in Europe. Many pilot projects are developed aiming to establish new recycling methods, mostly based on a chemistry approach. However, only a few of them appear to be upscalable in the nearest future.

All described above processes are connected together tightly. Especially sorting is important to perform effective recycling. For example, for chemistry approaches the presence of some dyes, as e. g. Levafix Brilliant Red E-4BA, can be disturbing. Thus, textiles containing these dyes are not suitable for chemical recycling and should not be applied for it.

Notably, red color can be given to clothes with different chemicals and not all of them present a problem for define recycling strategies. This is a good example, how composition of clothes determines the optimal recycling method for it. Unfortunately, this is a point that is not clear to all clothes producing companies nowadays. More conscious sustainable design of textile materials could elevate the recycling success significantly.

Since in our project we aim to create a new recycling method only for cotton, polyester and blends of these materials, it would be necessary for us to find a partner providing us a textile waste stream with exactly these textiles. During further optimization of our process in the future, we might would have to define the desired feedstock even more detailed to eliminate the presence of disturbing factors. It will be of great significance to study the influence of non-textile components on our strategy. Additionally, cooperations with fashion industries would be helpful to elevate the overall sustainable development of the textile industry with our project.

After learning about the general recycling processes and its difficulties, we wanted to get in touch with fashion companies to get their point of view regarding the textile waste issue. We wanted to know what clothes are made from and why. We also wanted to know how recyclable fashion is designed and what problems can occur when it comes to recycling and selling of the recycled clothes.

We contacted a lot of brands to find out about their approach to sustainable fashion. Unfortunately, this was much harder than we first thought as many brands were reluctant to talk to us and did not respond to our requests for an interview or only referred us to their websites and sustainability reports. Nevertheless, we were very happy to find some companies which were willing to share their vision of sustainability and their unique approach to the issue. This helped us to understand how the future design of textile products could look like and how it could facilitate the recycling of clothes.

We were able to talk to German retailer Otto about their circular collection, as well as Cotton n More and Rifo Lab, both companies that sell clothes and try to make the world a little more sustainable. From these companies we learned about interesting and unique approaches to sustainable fashion in all its forms. From collecting clothes for recycling, to designing a recyclable collection, to the different certificates that indicate a more environmentally or socially sustainable product.

Following our discussions with textile manufacturers, we also wanted to get in touch with companies that resell used clothing to gain a deeper insight into the strategies for textile waste reuse. We contacted a number of companies and organizations to do this, however, not many of them responded to our emails or phone calls. So we are really grateful that we were able to talk to Familienleben e.V. Dresden.

Expert talk with OTTO

OTTO is a large German online shop and fashion brand that has taken an honorable approach to making its clothes more sustainable. They offer what they call a “circular collection”, which is designed to be easier to recycle and is part of OTTO’s strategy to offer a wider range of sustainable products by 2050.

During our conversation with OTTO, we learned that designing a sustainable clothes collection is not a trivial task. Traditional garment design has to be critically rethought, and alternatives have to be developed for some garment elements. For example, it is necessary to make buttons and rivets removable, which OTTO has achieved by adding a twist lock to this detail.

Another interesting feature developed by OTTO for the circular collection in cooperation with circular.fashion GmbH is the circularity.ID®. This is an NFC tag or QR code integrated into the garment which, when scanned, provides access to information on the composition of the garment. The circularity.ID® also gives consumers the knowledge of where to take their old clothes for recycling or reuse. Such product identifiers should make the fashion industry more transparent and facilitate the recycling and reuse of textiles.

Nevertheless, it is important to note that the distribution of sustainable products remains challenging, as they are typically more expensive than traditional clothing. This task could be even more difficult for unestablished brands. Therefore, well thought-out marketing strategies or cooperation with more experienced companies would be of great importance to sell sustainable products.

As we discussed with the representative of OTTO our project, we were advised to explain well our method to a broader public, whereby underlying the applied safety concepts. A lot of people still associate genetically modified organisms with something dangerous and “bad”. Therefore, when products made from our monomers are brought to market, it would be important to remain transparent about our technology and educate the public about synthetic biology.

Expert talk with Cotton n more

Cotton n more is a fashion company specializing in the production of cooperative fashion and merchandise. The brand is committed to sustainability, using renewed or recycled materials wherever possible. It also holds several social and environmental certifications, including the International Recycling Standard (GOTS) and the Green Button. These certificates represent that cotton n more is both interested in the well-being of the environment as well as in the well-being of the employees. The certifications are awarded for different areas and require different standards of social or environmental sustainability. For example, the GOTS requires that no child labour is used and that living wages are paid, but it also defines global organic standards. The Green Button certificate also defines environmental and social standards. Cotton n more also has the Global Recycling Label, which certifies that the materials used have been recycled under good working conditions and using ecologically sound methods. The label also sets limits for chemicals and requires customers and other stakeholders to be able to trace the labelled product.

During our communication with the company, we wanted to learn more about their specific use of recycled materials and the textile composition preferred by their customers. In our discussion we discovered that around 50% of their clothing is made from pure cotton and that recycled PET from used PET bottles is utilized in blended textiles, whenever possible. Cotton n more told us that not all of their customers order products containing the recycled PET, but that there is a general interest in recycled materials. We have also learned that in order to comply with certain labeling standards (GOTS), 70-94 per cent cotton has to be present in the final product, so that the polyester amount is kept low. The company also made us aware of the environmental threat posed by dyes, which needs to be addressed, especially in textile-producing countries, and could be a starting point for another iGEM project to make the fashion industry more sustainable.

Cotton n more said that our project ReFiBa does not seem to violate the guidelines of any sustainability certifications or environmental labels. They, thus, mentioned, that they could imagine working with ReFiBa, even if genetically modified bacteria are applied. In fact, Cotton n more were already working with a company that uses bacteria to decompose PET bags. The many labels the company has acquired showed us how companies can be motivated to become more sustainable in general. When an effective method for recycling would be established, networking with organizations developing such certificates could be very beneficial to evaluate the significance of the method and to promote its application, resulting in an overall bigger impact on the world.

Expert talk with Rifo

On our journey to find fashion brands that already use recycled textiles in their products, we also learned about Rifo Lab. Rifo Lab is an Italian brand that has a special concept for collecting used clothes. This brand offers to have a courier service collect at least 5 items of clothing from customers and take them to a recycler. Customers can also drop off used clothes at a local store and receive a voucher for the Rifo lab products.

The collected clothes are sorted by hand and filtered for clothes made of mono-materials, because only these textiles can be recycled and made into new clothes by Rifo lab. Parts of the materials which cannot be recycled, Rifo Lab downcycles to produce “fluffy packs”. Fluffy packs are shipping bags made from textile waste which can also be reused later by customers, e.g. as cosmetic bags. Production of fluffy packs is another example how new value can be given to textile waste. Additionally, the use of such shipping bags helps reduce plastic consumption.

As we have discussed our project with the owner of the company Niccolò Cipriani, he told us that our project would have to be economically profitable and scalable to an industrial level in order to be implemented in the real world. According to Mr. Cipriani, the use of GMOs in the process should not result in a problem with the distribution of the resulted products. Furthermore, Mr. Cipriani underlined the importance of the logistic concepts in the recycling strategies.

Expert talk with Kleiderkammer

An alternative to recycling clothes is to sell them on or to donate them so that they can be used again. To find out more about this second-hand approach, we spoke to Familienleben e.V., who run a clothes chamber. In particular, they focus on clothing for children and mothers. Their system is based on the fact that anyone can choose clothes from the chamber and take them away in return for a small donation to maintain the facility. According to a survey conducted by Familienleben e.V., the main reason why customers come to the cloth chamber instead of buying new clothes is that they want to live more sustainably.
From our discussion we learned that clothes are coming to the clothes chamber as donations from other people. The received clothes are sorted by hand by volunteers. The charity can use about 75% of the donations, the other 25% cannot be reused and have to be discarded. Clothes that cannot be reused by the organization are given to the German Red Cross, which organizes the further disposal or recycling of textiles.
Finally, we found that there is not much of a competition in the secondhand textile market. Additionally, since the amount of given-away or discarded clothes is very high and not all garments can be reused, the presence of an established recycling strategy would not negatively impact the secondhand community. In fact, it would provide them with an alternative for what they could do with the received clothes not suitable for their purposes.

Data Workshop

Before we took the first crucial steps—such as assigning specific responsibilities, finalizing our ideas, and forming our team—we wanted to establish a solid foundation for our data management. To achieve this, we consulted Dr. Susann Auer, based on Dr. Kai Ostermann's recommendation. She joined our meeting on February 13th and gave a presentation on how to structure and store our data effectively. We learned how to use TU Dresden’s cloud storage, where our project files are now securely stored. The data management system we implemented was heavily influenced by her advice and proved invaluable throughout the project. During her presentation, we had the opportunity to ask her questions about team responsibilities and organizational structure. Interestingly, the structure we had initially devised was quite similar to her recommendations, allowing us to optimize it further. This resulted in a well-organized team with several leaders, a project manager, and subgroups dedicated to tasks like the wiki, funding, and more. Our data is stored on multiple servers with several months' worth of backups, ensuring that all our work is secure.

Biological Safety

The question of how our project will be used in future applications is a key concern for many of those to whom we have presented our work. To evaluate both the safety aspects and potential future applications, we consulted various experts and biosafety officers. Our first discussion was with our supervisor, Prof. Thorsten Mascher, a B. subtilis expert, who provided insight into the safety considerations of our project.
Additionally, we met with Dr. Ulrike Scholz and Johannes Kautz, who are responsible for biological safety at our university, to further discuss relevant safety aspects of our project and its future use. Although Dr. Udo Mücke, the safety officer for genetic engineering in Saxony, was unable to attend due to illness, he kindly answered our remaining questions afterwards. For more details on the discussed safety aspects of our project, please visit our Safety page.

Public Outreach

Science aims to address challenges that affect us all. However, the full potential of scientific discoveries can only be realized when they are shared with and understood by the public. Engaging with the broader community ensures that the knowledge we generate is accessible, relevant, and beneficial to society as a whole. Our project tackles issues that have real-world implications, making it essential to communicate our findings and ideas to the public. With this, we decided to conduct a public survey about the impact of fast fashion.

Our Survey

To gain a better understanding of public opinion on textile recycling, we conducted a survey to assess how urgently the issue of textile waste is perceived. Collaborating with a psychology student, we aimed to ensure the survey was as representative as possible. From the 85 responses we received, it became clear that most people were aware of fast fashion and viewed textile waste as a significant problem, with an average rating of 4.1 out of 5 (where 1 indicates 'not a problem' and 5 indicates 'very problematic').

The survey revealed that while most participants care about the materials used in clothing, only 45.7% were aware that the majority of textiles are made from mixed fibers, and 27.1% mistakenly believed that natural fibers are the primary component in most clothing.
When it comes to the disposal of clothing, 60% of respondents said they donate clothes that are undamaged, and 27% even donate damaged clothing. Additionally, a large majority (92.1%) were aware that polyester takes a long time to decompose and knew that textile waste is often incinerated.

The vast majority of participants agreed on the importance of developing a recycling method for textiles and expressed minimal concerns about using genetically modified organisms (GMOs) to address this issue. The average concern level was 1.1 out of 5 (with 0 indicating no concerns and 5 indicating high concerns), with the primary worry being the potential for GMOs to enter the natural environment. Notably, 97.1% of respondents indicated that they would be willing to recycle their textiles if GMOs were used in the process.

Bielefeld European Meetup

From the 24th to the 26th of may we had the first opportunity to meet and connect with other iGEM teams competing this year. The BFH (Bielefeld-Frankfurt-Hamburg) organised their first meet up and 6 members of our team attended to represent us. Not only did we get to present our own project idea to other teams, we got to hear all about the interesting topics chosen by other contestants as well. There were workshops on a wide range of topics including how to make a promotion video, how to design your wiki, how to manage your data and many more. We were able to sit in on scientific talks, watch panel discussions with people heavily involved with iGEM and take part in a poster presentation, where every competing team could take the time to answer or ask any questions about their chosen project. We even got valuable feedback from current iGEM judges so we could be as prepared as possible for the grand jamboree in Paris.

Collaboration with iGEM Team 'KlothY' from Düsseldorf

During our project we have focused on the degradation of textile waste. However, in order to establish a closed recycling loop in the future, the chemicals obtained from our process need to be further converted into new products. This could be achieved, for example, by the method, which is currently being developed by the iGEM team “KlothY” from Duesseldorf. Their project mainly focuses on the genetic engineering of microorganisms to produce cellulose mats for textile production.
As our two methods complement each other, we decided to start a collaboration. Our idea was to degrade the cellulose mats produced by the iGEM team Duesseldorf into oligosaccharides and glucose, which they could then use to synthesize new cellulosic materials again. Unfortunately, due to time constraints, we have not yet been able to conduct all the planned experiments yet and the results of our collaboration will probably be available after the wiki freeze. If successful, our collaboration will be an important step in the development of a new circular economy concept.

Conclusion and Outlook

A lot of time has passed since the beginning of our project. Although we have followed a direction throughout our journey, our exact vision for textile waste management has been changing drastically almost every week as we have learned more and more about the field we are working in. Recognizing the limitations of our project, we invite the iGEM community and the rest of the world to join us in developing a sustainable textile and fashion industry. We invite you to think of waste as a valuable resource.
Furthermore, we invite you to think outside the box. Finding a better solution is a never-ending process. However, to achieve our goals, we need to do it together. The easiest way to influence the textile waste issue is to become more aware of our textile consumption and its impact on the world. In addition, a better exchange of information between clothes producing and distributing organizations and recycling companies is crucial to facilitate the recycling methods developed.

During our human practices activities, we have learned that it is important to:
- REDUCE the amount of textile waste.

At present, the volume of textile waste produced is too large to be effectively managed by any of the available recycling technologies. Thus, it is essential to limit the excessive consumption of clothes.
- REUSE old unwanted clothing.

Another way to minimize textile waste is to give old, unwanted clothes a second life. This can be achieved by e.g., donating or reselling old clothes.
- 'ReFiBa' those textiles that end up in textile waste and cannot be reused.

There are many communities that are trying to establish concepts for reusing clothes. However, when textiles lose their quality, they need to be recycled. Currently, especially the recycling of blended materials remains a huge challenge. It is therefore very important to develop a method for recycling mixed textiles. Exploring innovative recycling solutions to create alternative products, rather than just focusing on turning old clothes into new ones, could be highly beneficial given the substantial amount of existing textile waste.

With our project we have taken the first steps in this direction. The initial discussions with experts have helped us to refine the design of our system. However, we still need to carry out proof-of-principle experiments to show that it really works. Next, we need to assess how efficient and economical our solution is. Finally, we will need to contact various organizations to implement our method in the real world. For example, we will have to find someone who can sort textile waste with a defined composition for us. It will also be necessary to establish cooperation with companies that can use the products of our process.

In the future, we will continue to be transparent about the use of GMOs in our process. Although our survey shows that most people do not have a problem with the use of GMOs in textile recycling, this aspect still needs to be better explained to the general public. In addition, we need to further improve the safety concepts for managing the risks associated with GMOs and to present them to the people who want to use our products. Concluding, there is still a lot of work to be done to implement our project in the real world. This includes networking with various stakeholders to upscale our process in case further experiments with real textile probes prove the efficiency of our method.

References