Overview
Our project builds on a growing body of iGEM work that has sought to bring new solutions to the environmental impact of textile waste. While structuring our project, we drew inspiration from several previous iGEM teams that explored different strategies. For instance, Chalmers-Gothenburg 2020 designed an enzyme cocktail in E. coli for elastane degradation; Edinburgh 2021 immobilized cellulases on silica beads; and Greatbay SCIE 2022 investigated surface display systems for PETases and cellulases.
In particular, we took key inspirations from the 2024 TU-Dresden team for our approach. Unlike most previous teams, they utilized Bacillus subtilis as their expression host instead of the commonly used E. coli, highlighting its advantages for textile-degrading enzymes. They successfully cloned three types of cellulases: endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.176), and β-glucosidase (EC 3.2.1.21). We drew especially on the principles behind β-glucosidase and endoglucanase activity, integrating them into our design framework. Moreover, TU-Dresden demonstrated the significance of NaOH-autoclaved pretreatment for cotton/PET blend textiles, which became a crucial part of our degradation pipeline. This pretreatment lowers crystallinity and creates pores in the fibers, providing a more efficient pathway for enzymatic degradation.
While TU-Dresden successfully demonstrated spore surface display by fusing enzymes to CotY, we built upon their foundation and extended these ideas in our 2025 project. Under the title “Enzymatic Degradation of Mixed Cotton-Polyester Textile Waste: A Biocatalytic Approach for iGEM,” we focused on designing and constructing novel TfCut2 enzymes and six of its variants. Combining these with our engineered exoglucanase (1,4-β-cellobiosidase, EC 3.2.1.176) and NaOH pretreatment, we achieved more efficient depolymerization of cotton-polyester blends, enhancing fiber accessibility for enzymatic degradation. Unlike TU-Dresden, we expressed our enzymes in E. coli and optimized conditions to enhance activity on blended textiles, paving the way for a complementary strategy in enzymatic textile degradation.
Parts Collection
BBa_25JE6FNZ
TfCut2_5ZOA (wild-type TfCut2):
We contributed a new part for the wild-type TfCut2 (TfCut2_5ZOA), a
cutinase enzyme from Thermobifida fusca. Cutinases are
serine esterases that hydrolyze ester bonds, originally known for
degrading cutin in plant cuticles. TfCut2, however, has also been
shown to act on synthetic polyesters such as polyethylene
terephthalate (PET). By cleaving the ester linkages in PET, TfCut2
releases monomers like terephthalic acid (TPA) and ethylene glycol
(EG), which can then be recovered and reused. We successfully cloned
and expressed the wild-type TfCut2 into
Escherichia coli strains of DH5α and BL21 (DE3). Protein
expression was induced with IPTG and confirmed through SDS-PAGE and
Western blotting. This standardized part allows future iGEM teams to
explore enzymatic PET degradation and recycling pathways.
BBa_25GGFEQS
TfCut2_Variant1 (G62A/P193H/S197D):
In addition, we constructed six variants of TfCut2 to evaluate the
effects of rational and computational mutations on enzyme activity
and stability. Variant 1 introduces mutations at the catalytic triad
(H193, S194, D197) along with G62A, a mutation known to decrease
MHET binding and potentially shift substrate preference. We cloned
and expressed this variant in E. coli DH5α and BL21(DE3).
Expression was confirmed via SDS-PAGE and Western blotting. This
variant provides a starting point for studying altered catalytic
activity compared to wild-type TfCut2.
BBa_25ZBXUY9
TfCut2_Variant2 (H77R/D204C/E253C):
The mutations H77R, D204C, and E253C were chosen for their
functional effects on enzyme performance. H77R has been shown to
exhibit one of the highest overall binding affinities for both PET
and PBAT. D204C and E253C form a disulfide bridge that strengthens
ionic bonding and enhances thermal stability. Together, these
substitutions were designed to improve both substrate binding and
overall enzyme robustness.
BBa_250E9UVZ
TfCut2_Variant3 (A65H/L90A/I213S):
The mutations A65H, L90A, and I213S were selected to enhance
catalytic activity by building on known functional insights. A65H
may exert effects similar to the well-characterized G62A mutation,
which reduces MHET binding, while the combination G62A/I213S has
previously been shown to increase catalytic efficiency. Both L90A
and I213S are mutations derived from LCC, a highly effective
PET-degrading cutinase isolated from compost. By incorporating
naturally compatible LCC-inspired substitutions, this design
leverages the proven functional advantages of LCC to guide
improvements in TfCut2.
BBa_25JH17Z6
TfCut2_Variant 4 (D12S):
Variant 4 carries a point mutation by changing aspartic acid to
serine at position 12 (D12S). This mutation shows the highest
average log₂ fold change among non-conserved residues, indicating
improved enzyme stability without interfering with conserved
catalytic residues. While its stability improvement may be smaller
than Variant 5 (T234L), it carries significantly less risk of
disrupting enzymatic activity.
BBa_25WQ5BX1
TfCut2_Variant 5 (T234L):
Variant 5 introduces a threonine to leucine mutation at position 234
(T234L). This site was identified as the most stabilizing residue
substitution across all 261 positions. However, because this residue
is conserved, there is a potential risk of reduced enzyme activity.
This variant was designed to evaluate whether the strongest
predicted stability mutation can maintain enzymatic function despite
the conservation risk.
BBa_25PC5VWY
TfCut2_Variant 6 (Multiple residue substitutions):
Variant 6 incorporates all predicted stabilizing mutations except
those located in conserved regions. This design aims to test whether
combining multiple stability-enhancing substitutions can
collectively improve thermostability and PET degradation efficiency
while still preserving the enzyme’s functional activity.
Mutagenesis Working Pipeline
In search of our novel mutants, drylab went through a rigorous working pipeline including both the machine learning approach and the rational design approach with protein structures. A balance of both approaches allows one to explore and analyze new mutants in a more comprehensive way, and we encourage future teams to extend this workflow with even more advanced tools and studies.
Software
Our software, FiberSense, which identifies textile fiber through an image, contributes to daily textile categorization. It can be used by non-industry people to substitute expensive textile sensors, as the software only requires a phone to identify the fiber. It can also be used in the process of textile recycling and degradation. For example, the front-end workers of a clothing drop box or textile recycling plant will need to first roughly categorize the textiles in order to make the recycling process easier. A lot of the time, though, the labels may be damaged or cut-off, making the fiber content unidentifiable, and the front-end workers do not have the expensive textile sensor to categorize. However, with a textile identifying software that works on the phone, the workers can easily and efficiently categorize the textiles, fastening up the recycling process.
TfCut Mutant Library
Our TfCut2 Mutant Library combines literature-reviewed mutations with novel variants we developed through mutagenesis and computational approaches such as MSA, ASM, SSM, molecular docking, and MutCompute. By gathering both established and newly designed mutants, we aim to provide a resource that advances enzymatic plastic degradation research, supports sustainable solutions for the textile industry, and inspires future iGEM teams to explore TfCut2. Given TfCut2's well-documented reliability and potential to degrade plastics like PET and PBAT, our variants were specifically designed to enhance thermostability, reduce product inhibition, and increase weight loss efficiency. Through this contribution, we hope to strengthen the community's toolkit and open new possibilities for engineering next-generation polyester hydrolases.
GEMS Playground
To support the iGEM community and future teams, we created GEMS Playground, a collection of reusable educational and communication tools that demonstrate how synthetic biology and sustainability can be taught through media, games, and interactive design. These resources combine creative storytelling with scientific content, providing adaptable models for other teams to use in their own Human Practices, Education, or Communication work.
Podcast: Harry and Sydney and the Cell Next Door
Our podcast series offers a replicable model for science communication that blends fiction, storytelling, and synthetic biology. By showing how to structure episodes around real-world topics like plastic waste and enzyme design, we provide a framework future teams can follow to make their own projects engaging for non-experts. The scripts, tone, and structure demonstrate how to make complex biology approachable through conversation and narrative.
Audiobook: 《小魚逃亡記：微塑膠風波》 (Escape of the Little Fish: The Microplastic Crisis)
This audiobook contributes a new educational format for iGEM outreach (science explained through storytelling). It demonstrates how to communicate molecular biology concepts, such as enzymatic plastic degradation, to young audiences using accessible language and character-driven plots. By publishing it through an established educational platform, we also model inclusive communication methods that future teams can adopt to reach visually impaired or younger learners.
Digital Games
We developed five browser-based games: Textile Fighter, Textile Degradation Clicker, Enzyme Defense, Plastic Cutter, and Textile Sorter, as templates for interactive education in synthetic biology. Each game links biological logic to intuitive gameplay, demonstrating how coding and design can be used to communicate lab concepts. The open-ended structures and mechanics can be adapted by future teams to gamify their own topics.
SynBio Mahjong: Genetic Match
Genetic Match transforms traditional Mahjong into a game for learning genetic circuit assembly. By incorporating promoters, RBS, CDS, and terminators into the rules, it offers a model for connecting cultural games to molecular biology concepts. The design not only demonstrates how gene assembly can be taught through familiar gameplay but also opens new possibilities for linking cultural traditions with science education. As a team from Taiwan, where Mahjong remains a shared part of social life, we hope this approach encourages future teams to creatively merge local culture with synthetic biology, making learning more relatable and engaging for their communities.
Card Game: ATCG Match
Our DNA pairing card game contributes a simple framework for translating molecular interactions into classroom games. The mechanics demonstrate how to simplify abstract molecular rules for early education. Future iGEM teams can reuse or adapt this idea to design interactive tools for explaining genetic logic, molecular binding, or other scientific concepts.
LINE Stickers
Our LINE stickers show how digital communication tools can promote synthetic biology awareness in everyday life. They illustrate how future teams can translate project themes into simple, shareable forms that encourage environmental responsibility.
Team Merchandise
Our stickers, pens, and patches serve as replicable outreach models for teams seeking to extend project visibility through creative design. They show how visual identity and take-home items can strengthen engagement and serve as reminders of synthetic biology projects.
GEMS Playground contributes to iGEM by offering a library of adaptable educational tools and communication formats, from digital media to physical games, that integrate biology, design, and accessibility. Each element provides an example of how future teams can creatively convey synthetic biology concepts while expanding the ways iGEM connects with diverse audiences.