Sustainable textile production represents a critical challenge in synthetic biology, with bacterial cellulose emerging as a promising eco-friendly alternative. However, existing methods rely on multi-step processes that compromise efficiency and sustainability. In 2025, NAU-CHINA developed an integrated one-step weaving-dyeing system in E. coli, enabled by temperature-responsive genetic circuits and characterized through computational modeling and experimental validation. We are pleased to share our well-documented biological tools, engineered components, and implementation frameworks to support future iGEM teams in advancing sustainable fashion solutions. In brief, our contributions are organized into three key areas: Wet Lab, Model, and Human Practices.

In this year's contribution, wet lab cloned the gfaspurple into the pET-28a(+) vector to expand the functional characterization of the existing part BBa_K5357010 through comprehensive analysis of its thermal resistance and redox tolerance. The results have been documented in detail in BBa_25UIP3DW.

Purpose & Design

Non-fluorescent colored proteins can present clear and visible colors under ambient light, which gives them an advantage over fluorescent proteins in biomarking applications^[1]. GfasPurple is a highly colored protein-only chromoproteins^[2]. To extensively assess its robustness under challenging conditions, we conducted further research on this non-fluorescent purple protein in the Wet Lab section. We assembled BBa_K1033919 into pET-28a(+) for T7-regulated expression, proceeding with plasmid propagation in E. coli DH5α and protein production in BL21(DE3). Since H₂O₂ and Vitamin C are common biological redox species known to affect chromoprotein coloration, and because chromoproteins exhibit greater tolerance to high temperatures and redox species compared to fluorescent proteins, we extracted GfasPurple from bacteria and determined the effects of temperature, H₂O₂ and Vitamin C on the activity of GfasPurple to confirm this.

Figure 1. The plasmid map of pET-28a(+)-gfaspurple.

Result

Heat Tolerance Capabilities of GfasPurple: Our study evaluated the effects of different temperature treatments on the color stability of the Gfaspurple chromoprotein. The results demonstrated that GfasPurple could be maintained at 50°C for 60 min without significant color loss. When the temperature was increased to 60°C and 70°C, the absorbance at 579 nm (excitation wavelength) reached a peak, a phenomenon that might be attributed to increased scattering due to protein denaturation. At higher temperatures, the protein gradually lost its color and eventually formed an almost white precipitate at 90°C (Figure 2). In summary, GfasPurple was able to retain its characteristic purple color at temperatures slightly above room temperature, but exhibited poor stability under extremely high-temperature conditions, such as 90°C or greater.

Figure 2. Variation of absorbance of the sample under different temperature treatments.

H₂O₂ and Vitamin C Tolerance Capabilities of GfasPurple: To evaluate the stability of GfasPurple under oxidative and reductive conditions, we treated the crude protein extract with a gradient of H₂O₂ and Vitamin C concentrations and measured the resulting absorbance changes. Regression analysis of triplicate experiments showed that all data points aligned closely with the fitted curve, and the near-zero slopes of both regression lines indicated minimal change in absorbance across all tested concentrations (Figure 3 and 4). These results demonstrated that GfasPurple maintained stable coloration in the presence of varying levels of H₂O₂ or Vitamin C, confirming its strong tolerance to redox-active environmental factors.

Figure 3. Variation of absorbance of the sample under different H₂O₂ concentration treatments.
The dark red and light red areas represent the 95% confidence interval and 95% prediction interval, respectively. The red line indicates the regression curve fitted to the mean values from three independent replicates. The regression equation and coefficient of determination (R²) are provided in the upper left corner.

Figure 4. Variation of absorbance of the sample under different VC concentration treatments.
The dark blue and light blue areas represent the 95% confidence interval and 95% prediction interval, respectively. The red line indicates the regression curve fitted to the mean values from three independent replicates. The regression equation and coefficient of determination (R²) are provided in the upper left corner.

Our experimental validation confirmed GfasPurple's robust stability under both thermal and redox stresses, supporting its use as a sustainable bio-pigment across multiple fields. This chromoprotein maintained color integrity within defined temperature limits and exhibited strong resistance to H₂O₂ and Vitamin C, making it suitable for applications in food coloring, cosmetics, and textile dyeing. Furthermore, its cofactor-free nature and environmental compatibility positioned it as an ideal visual reporter for biosensors and diagnostic reagents in synthetic biology systems.

We believe that our team's systematic characterization of GfasPurple's stability under various temperature and redox conditions has provided valuable experimental data and methodological references for future iGEM teams working with chromoproteins. The quantitative tolerance boundaries we established offered practical guidance for selecting and applying chromoproteins in biosensors, bioreporters, or industrial dyeing projects, accelerating the development of robust visual systems in synthetic biology.

References

• [1]Sun G, Zha C, Su J, et al. Colored proteins act as biocolorants in Escherichia coli[J]. Molecules, 2025, 30(3): 432.
• [2]Liljeruhm J, Funk S K, Tietscher S, et al. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology[J]. Journal of Biological Engineering, 2018, 12(1): 8.

In the Model section of NAU-CHINA, the model group proactively communicates and collaborates with other groups, clarifying the requirements for verification, prediction, and innovative interactive promotion, and conducts practices supported by mathematical theories and computer technology. Our contributions include the following parts:

We have developed a predictable and customizable FourU temperature prediction model, which has solved the problem of regulation using temperature-sensitive RNA switches. This model integrated the thermodynamic parameters of Turner 99, quantified calculation of translation potential, and correction of ion concentration , and successfully predicted the decoupling temperature of this project. More importantly, our model can predict the switching temperature in other sequences or environments by changing key parameters in the model, such as GC content and ion concentration.

In addition, we applied the Kabsch algorithm, a classic structural alignment tool, to the design verification of fusion proteins, providing a quantitative and reliable solution to the key issue of whether the functional domain can maintain the correct three-dimensional structure after fusion. When other teams are conducting multi-domain fusion design, they can consider using this method to quickly assess the rationality of their concepts before experiments, significantly reducing the risk of functional loss due to structural damage and enhancing the success rate and efficiency of fusion protein design.

In the mathematical modeling section, we have also developed a complete set of mathematical models for microbial co-culture and open-source software, providing a solution for analyzing the interaction types of multi-strain collaborative systems. This model combines statistical hypothesis testing, Lotka-Volterra dynamics and grid search optimization, and can accurately quantify the competitive, neutral or reciprocal relationships among strains. We further transformed this complex theoretical model into an interactive desktop application based on PyQt5, enabling teams without a deep mathematical background to easily upload experimental data and obtain comprehensive interaction analysis with just one click.

We also designed a lightweight CNN training scheme for direction recognition and integrated it with bone and water-saving data to construct a dynamic size intelligent matching model with multi-level scoring and visualize the water-saving effect of this project, transforming the material advantages of synthetic biology into personalized experiences that the public can intuitively perceive.

Learn more about model

In the HP section of NAU-CHINA, we have actively worked to provide valuable resources and tools for future iGEM teams, building upon existing knowledge and promoting cross-team collaboration. Our contributions include several key activities:

Educational Outreach

We developed and shared a middle school series of popular science videos with the theme “From Nature, For Nurture”, with one episode focusing on bacterial cellulose fabrics. Together with the HKU-HongKong team, we co-created a bilingual popular science handbook on E. coli, addressing common public misconceptions and making knowledge more accessible. Additionally, we created a series of popular science posts titled “The Use of Synthetic Biology in Clothing, Food, Housing, Transportation”, further expanding public understanding of synthetic biology.

Please visit the education page

Collaborative Platforms

By developing the ICII web platform, we provided a space for long-term cooperation and content sharing. The platform served as a digital hub for iGEM teams from different regions to present and exchange knowledge, fostering cross-regional and cross-cultural collaboration and promoting the integration of global science and culture.

Please visit the collaborations page