On April 22, 2024, we celebrated the 55th World Earth Day with the theme "Global Plastics Battle". This year is also the 60th anniversary of diplomatic relations between China and France. During this time, important topics such as green energy, climate change, and biodiversity have been frequently discussed. The consumption of plastic products continues to rise each year, leading to a significant increase in plastic pollution, which has become one of the most urgent environmental threats facing the world. People are increasingly realizing that the issue of plastic pollution is much more complex than previously imagined. There is an urgent need for solutions to plastic pollution.

Do you know about plastic pollution?

(The data came from 400 randomly selected people)

Fig. 1   Partial results of questionnaire survey

Polyethylene terephthalate (PET), one of the most common plastics, gradually breaks down into microplastics in the natural environment. These are plastic fragments smaller than 5 mm and are difficult to degrade. These PET microplastics can serve as carriers for harmful substances, further exacerbating marine pollution and impacting entire ecosystems through the food chain. In 2023, researchers from Capital Medical University in China discovered the presence of microplastics in organs, specifically the heart and surrounding tissues, that had no contact with the external environment.

Fig. 2   The destruction of ecology of PET

Therefore, addressing the degradation of PET microplastics is urgent. So our team focuses on the degradation of PET microplastics in aquatic environments, aiming to provide practical applications and tackle real-world challenges.

To gain a deeper understanding of the current state of PET pollution, we consulted Professor Cheng Zhang, the Associate Director of the Department of Environmental Science at Jiangnan University. He is also a member of the "Ecological Think Tank" in Binhu District, Wuxi, and a member of the Chinese Society of Environmental Sciences. His research primarily focuses on the ecological and environmental toxicity of contaminants.

Fig. 3   Jiangnan-China members with Professor Zhang

Fig. 4   School of Environment & Ecology, JNU

During the discussion with Professor Zhang, we have gained a comprehensive understanding of the mechanism and current status of PET microplastic contamination. Professor Zhang explained that microplastics can harm human health by releasing phthalates and other substances. Studies have estimated that the amount of microplastics entering the ocean yearly ranges from 0.8 to 3 million tons. Plastic pollution spreads rapidly, with widespread impact, making global coordinated action crucial. This has further strengthened our determination to address the issue of PET microplastic pollution.

Moreover, Professor Zhang's proficiency in projects closely associated with environmental protection enterprises offered us valuable insights. He provided a comprehensive overview of the PET microplastic degradation industry, highlighting its potential for significant growth in the upcoming decades. Given the escalating environmental pressures, there are valuable opportunities for PET microplastic degradation technologies, indicating a promising future.

Fig. 5   Professor Zhang gave a vivid explanation of plastic bottles

Additionally, our literature review revealed various methods for microplastic degradation, including photodegradation, thermal degradation, and enzymatic degradation. Among these, enzymatic degradation involves the enzymatic breakdown of PET, resulting in no potentially harmful residues, making it more environmentally friendly, efficient, and sustainable. In 2016, a novel PET hydrolase called IsPETase from Ideonella sakaiensis 201-F6 was discovered ^[1]. So far, IsPETase is the true PET-degrading enzyme produced by natural evolution.

However, due to PET microplastics' hydrophobic nature, wide distribution, and low concentration, the interaction between PETase and microplastics is significantly hindered, negatively affecting the degradation efficiency of PETase. Our further literature review discovered a class of short polypeptides known as plastic-binding peptides, which can bind to plastic surfaces through hydrophobic interactions and other forces. These peptides represent a potential solution to enhance PETase's capability to degrade PET plastics. This inspired us to explore the use of synthetic biology techniques to identify binding peptides with high affinity for PET and to construct PETase-PET-binding peptides to tackle this challenge. This approach could significantly provide a novel solution for addressing the pollution of PET microplastics.

In addition to gathering background knowledge from academia, we also established connections with government agencies and environmental enterprises, including the Wuxi Environmental Protection Agency and Wuxi City Environmental Technology Co., Ltd. Through our contact with Wuxi Environmental Protection Agency, we gained valuable insights into the current plastic management situation in Wuxi and the policies being implemented to reduce PET microplastics, deepening our understanding of the project background.

Ms. Yu, the staff member of Wuxi City Environmental Technology Co., Ltd., also guided us on a tour of Peach Blossom Mountain, a massive landfill site. She explained that our proposed solutions could effectively reduce secondary pollution to the environment compared to conventional landfilling and incineration. Her encouragement and affirmation undoubtedly infused our project team with significant confidence and motivation.

Fig. 6   Jiangnan-China discussed with the staff members of the Wuxi Environmental Protection Agency

Fig. 7   Jiangnan-China in Wuxi City Environmental Technology Co., Ltd.

Fig. 8   Jiangnan-China visited Wuxi City Environmental Technology Co., Ltd. exhibition hall

Fig. 9   Jiangnan-China experienced games about environmental knowledge

Fig. 10   Jiangnan-China stood on "Peach Blossom Mountain", a massive landfill site

Based on our preliminary research, our project aims to leverage synthetic biology techniques to identify PET-binding peptides and construct PETase-binding peptide fusion proteins to address the pollution of PET microplastics. However, traditional biological experiments for screening high-affinity peptides face cumbersome experimental procedures, time-consuming processes, and high labor costs, significantly limiting the application and promotion of PETase in the degradation of microplastics.

Consequently, we approached Professor Zhaohong Deng from the School of Artificial Intelligence and Computer Science at Jiangnan University. He is a pioneer and expert in bioinformatics and intelligent data mining and modeling.

Fig. 11   Professor Zhaohong Deng

Professor Deng introduced us to the immense potential of deep learning, particularly in synthetic biology. He explained how it can analyze vast amounts of biological data, design novel biomolecules, optimize genes and metabolic pathways, and even predict protein structures. This approach significantly reduces labor- and time-cost while enhancing biosynthetic efficiency. Therefore, we plan to utilize neural networks within deep learning to identify PET-binding peptides with a high affinity for plastics.

Fig. 12   Jiangnan-China members with Professor Deng

Fig. 13   School of Artificial Intelligence and Computer Science, JNU

Through brainstorming, we optimized our project design to focus on screening efficient PET-binding peptides using a deep-learning neural network prediction model. Our objective is to identify PET-binding peptides that effectively bind to PET microplastics. By leveraging deep learning techniques and synthetic biology, we aim to discover high-affinity PET-binding peptides and construct PETase-PET-binding peptide fusion protein to enhance the PET degradation efficiency. This approach will facilitate the application of PETase in the environmental degradation of PET microplastics.

Fig. 14   Team meeting

Furthermore, we propose a solution strategy, abbreviated as STORM, which means Screen, Test, Optimization, and Re-Modification.

Screen: Establishment of a one-dimensional Long Short-Term Memory (LSTM) model for initial screening and a three-dimensional Graph Convolutional Network (GCN) model for re-screening to screen efficient PET-binding peptides.

Test: Construction of fusion proteins by fusing peptides with the N-terminus and C-terminus of PETase, respectively, for validation and characterization.

Optimization: Optimization of fermentation conditions and linker to improve the expression of fusion proteins.

Re-Modification: Introducing beneficial mutations to PET-binding peptides to further improve the substrate binding efficiency as well as degradation efficiency of fusion proteins.

Fig. 15   Overall research strategy

As our project progressed and we identified several PET-binding peptides that effectively enhance PETase activity against PET microplastics, we met again with Professor Deng. Regarding our work using LSTM networks for amino acid sequence screening, he offered valuable suggestions: by introducing a multimodal voting mechanism or adjusting threshold strategies, we could further improve the accuracy and efficiency of our screening process. During the subsequent GCN re-screening phase, Professor Deng innovatively proposed using LSTM as a feature extraction tool to deepen our understanding of the characteristics of amino acid sequences, which is expected to enhance the predictive accuracy of the GCN model significantly. This idea broadened our perspective on feature engineering and provided a new viewpoint for interdisciplinary applications. Additionally, Professor Deng guided us to think critically about achieving precise data representation of the target peptides and explore the potential significant correlations between the target labels and the target peptides. He encouraged us to consider integrating a scoring mechanism into the model training objectives, aiming to optimize the training process and directly strengthen the significant correlation between the selected amino acid sequences and the target peptides, thereby enhancing the overall effectiveness of the screening system.

After achieving promising results in the laboratory with artificial simulation of PET microplastic degradation, our team conducted an in-depth field survey at Wuxi Yongda Wastewater Treatment Co., Ltd. We aimed to compare our project with the current biochemical treatment methods used at the wastewater treatment plant, to validate the beneficial effects of our approach on microplastic removal from water bodies.

General Manager Qing Xie of Wuxi Yongda Wastewater Treatment Co., Ltd. guided our team on a tour of the wastewater treatment facility, providing a detailed overview of the current biochemical methods used. Our team meticulously recorded the specific processes and important data such as treatment temperatures and pollutant concentrations. This information plays a crucial role in assessing the feasibility of our project's implementation.

Fig. 16   Wuxi Yongda Wastewater Treatment Co., Ltd.

Fig. 17   Mr. Xie shown us around the wastewater treatment facility

After our team presented the specific details of our project, we engaged in extensive discussions. The technical engineers at the wastewater treatment plant expressed strong interest in our initiative. They mentioned that the facility still faces challenges in effectively managing plastic fragments and microplastics. Applying synthetic biology methods to construct PETase-binding peptide fusion proteins for PET microplastic degradation could potentially provide a new extension to their wastewater treatment approach, effectively improving the cleanliness of treated water and promoting healthier, greener living for residents.

Fig. 18   Jiangnan-China discussed with the manager of the wastewater treatment facility

During the discussion, we gained insights into current environmental policies and inquired about the plant's views on our project. The facility expressed concern about whether our project would ensure environmentally safe disposal. In response, we provided a detailed explanation of the project's environmental safety design.

While they raised some questions about the project's short-term scalability and rapid promotion, they acknowledged the high level of innovation and practicality in our design. More importantly, they expressed confidence that our project would play a significant role in addressing microplastic pollution.

Fig. 19   Jiangnan-China with the manager Mr. Xie

Additionally, we submitted a brief project report to Associate Professor Guoqiang Xu from the School of Biotechnology at Jiangnan University. Professor Xu has extensive experience in synthetic biology and is involved in the "Synthetic Biological Systems and Bio-Manufacturing" international joint laboratory project and two industry collaboration projects. He has also guided teams that participated in two iGEM competitions.

Fig. 20   Associate Professor Xu directed Jiangnan-China

Associate Professor Xu shared his iGEM competition experience with us and provided numerous valuable suggestions for our project. His insights significantly contributed to the refinement and improvement of our work. We are very grateful for his patient guidance and detailed, constructive feedback. At the end of our conversation, Professor Xu expressed his confidence in the potential of our project and looked forward to our future progress.

Fig. 21   Jiangnan-China with Associate Professor Xu

Fig. 22   School of Biotechnology, JNU

After completing the project, we visited Jiangsu Zhongman Environmental Technology Co., Ltd. for promotion and communication. Manager Qianshan Yue introduced us to Zhongman's latest ecological protection and environmental governance achievements. We actively discussed the project's prospects and potential for future collaboration.

Fig. 23   Jiangsu Zhongman Environmental Technology Co., Ltd.

Manager Yue stated, "With the increasing societal focus on environmental issues and ongoing technological advancements, the plastic disposal sector is poised to become the next blue ocean market. The outcomes of this project are expected to achieve environmentally safe disposal of microplastic pollution, which not only supports the development of the plastic disposal industry but also contributes to environmental protection and pollution cleanup. This could become a new trend and potential opportunity in the current market." This perspective provided clear direction for our project and inspired our team members with boundless hopes and expectations for future development.

Fig. 24   Jiangnan-China discussed with Manager Yue

Fig. 25   Jiangnan-China with company personnel

In addition, we have re-established contact with Professor Zhang. He acknowledged our project and engaged in creative discussions regarding potential future applications with our team. In the future, we can also consider salvaging water cyanobacteria, and adding a "short peptide-linker-enzyme" fusion protein complex during processing, which can not only solve the problem of PET microplastic degradation, but also solve the eutrophication of water caused by cyanobacteria.

Afterward, we integrated human practices with education and communication in synthetic biology. We sought public opinions on our project through public outreach presentations, iGEM team exchanges, and forums. The general response was positive, and participants engaged in creative discussions with our team regarding potential future applications. For detailed information, please refer to the Education & Communication section.

Are you optimistic about our project?

(The data came from all respondents)

Fig. 26   Partial results of a questionnaire survey

It is particularly worth mentioning that, inspired by the Shanghai-Tech team last year, we also provided a brief agreement for stakeholders involved in our program.

Fig. 27   Disclaimer: All of the above information is provided with permission

[1] Yoshida S, Hiraga K, Takehana T, et al. A bacterium that degrades and assimilates poly(ethylene terephthalate) [J]. Science, 2016, 351(6278): 1196-1199.