Cold regions around the world include the Arctic, Antarctic, and high-altitude mountain areas. Areas with an annual average temperature below 5°C cover approximately 22% of the Earth's surface, while regions below 0°C account for 15%, severely impacting human production and daily life. Faced with this formidable challenge, our team feels a profound sense of responsibility and is determined to build this project into a “strong shield” against ice and snow.

After launching the project, the team swiftly initiated brainstorming sessions but soon hit a bottleneck in directional choices. Just as we were at a loss, inspiration struck from the ancient Chinese text Zhuangzi: “In the Northern Ocean swims a fish, its name is Kunpeng.” This mythical creature, said to traverse the frigid depths, prompted us to seek cold-resistance wisdom from nature.

We turned our gaze to the vast natural world, systematically researching survival strategies of various organisms against extreme cold. During this process, team members independently converged on a single keyword: antifreeze proteins. To further explore their potential, we visited Professor Zhang Lei, an expert in this field. From him, we learned about the remarkable thermal hysteresis activity and potent ability of antifreeze proteins to inhibit ice recrystallization—a discovery that captivated us. The team swiftly established collaboration with Professor Zhang and relayed this information to the wet lab group.

To address the core question of iGEM's Human Practices section—“How does our project influence the world around us? And how does the world around us influence our project?”—we innovatively developed a “protein secretion” model. Drawing inspiration from the stepwise processing of antifreeze proteins—from translation and modification to secretion—we proposed a corresponding tiered strategy: “society → education → experts → industry → government.” This approach enabled deep engagement with stakeholders across multiple levels.

During the track selection phase, we gathered multiple potential applications for antifreeze proteins through campus open days, including healthcare, protective coatings, and food security. Among these, a comment regarding “skin irritation in cold conditions” caught our attention. To validate this need, we interviewed cosmetic enthusiasts and experts, ultimately narrowing our project focus to the fashion skincare sector.

Society is the first choice for iGEM Tianjin 2025 team to promote our own project, and it is also an important way for us to interact with all sectors of society.

Within the iGEM Tianjin 2025 team, social interaction serves not only as the core driving force propelling our project forward but also as a vital bridge connecting us with diverse communities. Society functions much like the ribosomes within a cell—acting as a critical site of synthesis, it decodes the information (mRNA) conveyed by society and initiates the preliminary transformation of our project (protein).

Drawing inspiration from early engagements with diverse communities, we defined this year's project direction. Throughout development, we maintained multidimensional public interactions, continually listening, learning, and refining our work. Simultaneously, the team actively gave back to society through ongoing communication and outreach, spreading the positive impact of synthetic biology and inclusive aesthetics.

On April 5, 2025, the "Crabapple Blossom Festival" campus Open Day arrived as scheduled. More than 100,000 teachers, students, alumni, citizens and tourists gathered for this spring event integrating 130 years of history and culture with scientific and technological innovation.

The iGEM Tianjin team at this year's "Crabapple Blossom Festival" emphasized the comprehensive human practice model of promotion and interaction, which received a good response. The booth was exposed to more than 20,000 people, collected more than 500 effective survey results, and was promoted by the official WeChat account of Tianjin University.

During this Crabapple Festival event, we officially promoted and popularized the core concept of iGEM Tianjin 2025 - "Anti Freeze Protein" (AFP) to the public for the first time. Through innovative formats such as little snowman making and offline questionnaire surveys that are popular with the public, we enhanced awareness of Anti Freeze Proteins and solicited practical needs related to frost resistance. This year, iGEM Tianjin 2025 team innovatively designed creative activities like the "Nine-Nine Cold-Dispelling Chart" and "Crabapple Blooming on Branches", integrating HP information collection with traditional Chinese culture. While gathering public opinions on Anti Freeze Proteins, these efforts also promoted the inheritance and development of traditional Chinese culture.

Through analysis of the collected survey data, we identified significant public interest in frost damage prevention and cold protection solutions. While addressing conventional needs such as vehicle door insulation and road surface frost resistance, the survey also revealed innovative applications that sparked our curiosity. Notably, the demand for frost-resistant cosmetics presented a fresh perspective, which ultimately became one of the key inspirations for our project.

With the rapid development of AI, so many industries are trying to integrate AI technology with their own projects, while our team is no exception. This year, we tried to build an AI model for our own project, aiming to provide a more convenient, comprehensive and efficient way for the society to understand our project.

The AI model is mainly built and trained around our project, and can now answer questions related to the design details of this project, wet lab engineering implementation, experimental scheme and other aspects

Through this AI model, we hope to reduce the cost of human resources and convey the core concept of our project to all sectors of society through more targeted answers.

Meanwhile, we have established a standardized user feedback loop mechanism through AI models, continuously optimizing project content and model performance by interacting with users. While adhering to data security and privacy protection standards, we integrate suggestions from diverse users, significantly enhancing the AI model's responsiveness accuracy, service efficiency, and user satisfaction regarding our project content.

For details, please refer to the Model page.

Questionnaires are an important way for us to communicate and collect information with all sectors of society. This year, we designed a number of questionnaires from macroscopic to details, each of which has different functions and design details to make it more targeted and effective.

1.Information collection for the Crabapple Blossom Festival

During the Crabapple Blossom Festival and science education initiatives, our project was in its early stages. To address this, we developed more comprehensive and innovative research methods, including the previously mentioned "Nine-Nine Cold-Dispelling Chart" and "Crabapple Blossoms on Branches". While our survey revealed strong public demand for frost resistance, limited awareness of the functional benefits of frost-resistant proteins emerged. This realization highlighted the importance of subsequent educational outreach. Moreover, the cosmetics industry's frost resistance needs identified in public questionnaires provided fresh perspectives for understanding frost protection strategies.

2.Internationalized information collection

Considering that we hope to be able to put our project into more regions and countries, so that our project has a more international vision, we also designed a questionnaire for the global public, hoping to collect different opinions from various countries on the cosmetics users questionnaire.

In this foundational questionnaire, we first systematically engaged the public to gather nuanced insights into the tangible impacts of cold air exposure during autumn and winter seasons specifically focusing on skin health disruptions such as dryness, cracking, and sensitivity.

The data indicate that cold weather during autumn and winter exerts a notable influence on the skin of most individuals, so we further promote the follow-up research:

The initial data survey shows that although people's demand for autumn and winter skin care and anti-freezing is increasing, their awareness of anti-freezing protein is very little, which lays the foundation for our project and sets challenges for our follow-up popular science work.

3.Collection of information on users of cosmetics/skin care products

Throughout the entire project, we have consistently engaged in critical thinking and multidisciplinary research, which functions like the ribosomes within a cell, continuously synthesizing the core "functional components" that drive the entire project forward. By identifying issues through social investigations, refining strategies via consultations with academics and industry experts, and adjusting directions through global perspectives such as the Belt and Road Initiative, our ongoing research and reflection have served as the "template-guided synthesis process"—just as ribosomes follow mRNA templates to build proteins—ensuring the formation of practical solutions, the integrity of implementation frameworks, and the correct trajectory of the project from conception to execution.

Notably, some survey responses we collected highlighted cosmetic products' susceptibility to freezing and clumping during storage-a critical concern that aligned with our research objectives. Through further investigation, we confirmed this issue requires urgent attention, particularly in long-distance transportation and storage environments. Repeated freeze-thaw cycles under harsh conditions not only hinder product accessibility but may also damage active ingredients. Building on these findings, our project is developing innovative solutions by integrating antifreeze proteins into cosmetic formulas.

In addition, we also collected the public's views on biotechnology and genetic engineering, and found that people do have certain opinions about it, such as its safety and effectiveness. In order to ensure that our project is in line with bioethics and can be more convinced and accepted by the public, we further promoted the investigation in the subsequent Human Practices activities.

For details, please refer to the Government page.

As we move forward, the insights gained from these societal interactions will continue to inform our work in education, research, and industry collaboration, creating a truly responsive and responsible synthetic biology project.

Education is the first bridge between the project and social dialogue, and also the "endoplasmic network" in the "secretory protein release model" - it undertakes the initial processing and dissemination of the project concept, guides the public to understand the integration of synthetic biology and fashion aesthetics, and promotes project iteration in feedback. iGEM Tianjin hopes to popularize the knowledge of synthetic biology, expand the team's positive impact on society, explore the more far-reaching educational significance of iGEM outside the competition, and adopt the opinions of different groups of people to improve the project. We have mainly carried out systematic and multi-level science popularization and education activities for four groups: children, middle school students, college students and the public, aiming to break down knowledge barriers, stimulate scientific interest, and spread inclusive aesthetic concepts.

1.Popular Science Summer Camp: Jingui Ningming Practice Team

In July 2025, the Jingui Ningming Practice Team of Tianjin University came to the Third Primary School in Chengzhong Town, Ningming County, Guangxi Zhuang Autonomous Region, China to carry out a science popularization summer camp. Team members teach popular science courses on the basics of biology and lead children to understand with games.

2.Biology Knowledge Fun Game: Synthesis of Secretory Proteins

In order to solve the problem of children's insufficient knowledge reserve when popularizing complex content of science, we have designed an interesting biology game for children aged 6-12, and completed the test, modification and dissemination of the game at No. 3 Primary School in Zhongzhen, Ningming County. The game simplifies the process of secreting protein synthesis, in which children play organelles.

The following are the rules of the game:

1.Climbing the Peak Research Camp

In August 2025, nearly 100 high school students from all over China came to the School of Synthetic Biology and Biomanufacturing of Tianjin University for research. Wu Yi, the instructor of iGEM Tianjin, gave a popular science lecture on large fragment genome synthesis; Captain Wen Renkui introduced the project content of iGEM Tianjin2025. The students asked questions enthusiastically, showing their interest in promoting scientific content.

In the seminar camp, we also helped guide students to complete simple experiments on synthetic biology:

- Understand citronella alcohol and how to synthesize this low-natural and expensive perfume raw material by synthetic biological methods; then mix perfume with rose alcohol (L-citronella alcohol) and corn double dealdehyde alcohol, and conduct multi-dimensional sensory evaluations and write evaluation reports.

- Understand the principle, process and experimental device of pharmaceuticals; use a single stamping tablet machine to press the pharmaceutical tablet, then use electronic analysis balance to detect the weight of the pill, detect the quality of the tablet with a brittleness tester, and analyze the weight and quality of the drug according to the standard.

- Understand the morphology of the most common mycorrhiza and aspergillus, make tablets, and observe it with an optical microscope; use sterile suction heads to pick Escherichia coli bodies carrying different types of fluorescent proteins, mark them on the medium, and use flat marking techniques for microbial painting. After staying overnight, you can see the corresponding color pattern under the Blu-ray meter.

- Understand Bill's law and use a spectrophotometer to determine the anthocyanin content in dragon juice; learn redox reaction and use color reaction to determine the reductive vitamin C content in orange juice; and then use dragon juice and orange juice to make ice cream.

Under our help, the students not only learned more about the experimental knowledge of living things, but also improved their experimental skills and experienced the fun of experiments by doing it themselves. After that, we guided the students to complete the experimental report.

2.Science Popularization Open Day

In May 2025, the iGEM Tianjin 2025 team cooperated with the Bioscience Popularization Base of the Institute of Chemical Engineering to launch an interesting science popularization activity for high school students. This science popularization open day activity fully stimulates teenagers' interest in life exploration and synthetic biology by displaying, experiencing and educating the comprehensive human practice model of multi-block coordination. The creative card game conveys the mystery of genes between the square-inch cards; the intuitive and vivid perfume experience link makes synthetic biology clear and visible; and then to the interesting microbial painting exhibition, art and science are perfectly integrated in the pattern. Through the comprehensive application of different forms of science popularization, we aim to popularize the basic knowledge of synthetic biology, pull the juice between the public and cutting-edge research, and create a good atmosphere for the whole society to pay attention to and support scientific and technological innovation.

UNO：The mystery of gene operation

Each player chooses a common enzyme card and puts it on his side, facing up. At the same time, he takes out 7 cards from the pile as a hand, and takes out a DNA card from the pile and puts it on the table, facing up, as the initial sequence.

When the process is DNA replication on the spot:

Play the dNTP card, connect the phosphate diester bond/hydrogen bond, and the DNA polymerase participates in the replication process. The card is turned to the reverse side, and the DNA replication process continues at this time;

Play the NTP card, connect through hydrogen bonds, and the RNA polymerase participates in the transcription process. The card is turned to the reverse side, and then the extension process of mRNA is carried out.

When the on-the-sit process is an extension of mRNA:

The NTP card is connected through the phosphate diester bond, and the RNA polymerase participates in the transcription process. The card is turned to the reverse side, and the extension process of mRNA continues at this time;

In particular, if you can play three NTP cards that can be paired with the first three NTP cards for base complementarity according to the current mRNA extension sequence, these three NTP cards can be connected. DNA polymerase and RNA polymerase do not participate in the decoding process of ribosome, and there is no need to turn over. At this time Continue the extension process of mRNA.

During the game, you can use the corresponding function cards to make yourself win faster. The gene mutation added function card can force the next house to draw two cards; the gene mutation missing function card can skip the next round; the two-way copy function card can reverse the current sequence direction, and force subsequent players to play cards in reverse...

The first player to play the hand is the winner of the game. For more details of the game, please check the game rules book. ( Here is a detailed description of the rules of the game inserted in the hyperlink)

1. iGEM Team & Competition Briefing

In November 2024, iGEM Tianjin launched a new lecture. Wu Yi, the instructor of iGEM Tianjin, gave a popular science lecture on what synthetic biology is, what problems synthetic biology is solving, and what is the iGEM competition. Everyone can experience the charm of synthetic biology from shallow to deep.

The new captain, Renkui Wen, reviewed and summarized the iGEM Tianjin 2024 project. Focusing on the increasing challenges faced by human beings in exploring space, the iGEM Tianjin 2024 team designed an engineering yeast that can synthesize high-value compounds under different induction conditions using specific genetic circuits and metabolic pathways to build a life support system for space exploration and provide important new quality productivity using synthetic biology. Under blue light, the engineering yeast can efficiently produce melatonin to improve astronaut sleep, regulate mood and improve arrhythmia. In the presence of galactose, the engineering yeast can quickly synthesize saffronic acid to improve the oxygen utilization rate of astronauts and thus effectively improve work efficiency. Through this lecture, the participants had a deeper understanding of the iGEM competition in synthetic biology.

2. Conference of China iGEMer Community（CCIC）

In August 2025, the iGEM Tianjin 2025 team participated in the China Biogenetic Engineering Machine Exchange Conference in Beijing. At the conference, Jiang Shan, founder of red panda biology, Wu Yi, instructor of iGEM Tianjin, and other industry celebrities reported on the development process of synthetic biology, cutting-edge support technology of synthetic biology, DNA design and synthesis technology of genome fragments. The iGEM Tianjin 2025 team fully discussed the experimental scheme, technical details and industrialization feasibility through the exchange of opinions with other iGEMer projects in the communication session and road show display in the poster area, and the atmosphere of academic interaction on the spot was strong.

We disseminate synthetic biological knowledge, inclusive aesthetics and sustainable fashion concepts to the society through new media platforms and public content output.

1. Official account series content

We have operated two columns, "Synthetic Beauty" and "They are Watching", and released a total of 80 pushes, with more than 12,000 views. We systematically explain the application of synthetic biology in the field of cosmetics, and advocate breaking aesthetic biases and opposing gaze.

Please search "iGEM 天津" on Wechat to access our official account.

2. The integration of traditional culture and scientific narrative

The project logo is based on the "kun" in Zhuangzi's book Zhuangzi, an ancient Chinese philosopher. Kun lives in the cold area of the far north, which contains Zhuangzi's idea of "remoting out of vulgarity" and symbolizing the oriental wisdom of "bring all things and tolerating diversity". We enhance the public's identity with the project through cultural symbols, and also convey the innovative voice of the integration of China's science, technology and culture to the international stage.

In order to better connect synthetic biology with traditional Chinese culture, we have established a cooperation with NAU-CHINA. On the Into China, Into iGEM (ICII) platform established by NAU-CHINA, we shared the content and cultural insights about our project.

To visit the website, please click the link.：http://www.icii-nau.cn/

Our vision:

In the HP model, education is not only "output" but also "input". Every popular science activity, every dialogue and exchange brings us new inspiration and feedback, and promotes the continuous optimization of the project in the three dimensions of technology, aesthetics and communication. Just as the "endoplasmic network" in the secretory protein model, education allows the core concepts of the project to be initially modified and targeted, and also lays a cognitive foundation for the subsequent "industrial processing" and "government docking".

The Expert Module serves as a cornerstone for in-depth engagement with domain specialists, enabling the systematic integration of professional insights into project development. Experts from diverse fields function as conceptual "mitochondria," providing sustained intellectual energy and strategic direction that drive the efficient translation of societal needs into technically viable pathways. Through ongoing interdisciplinary exchanges, we continuously refine research objectives, identify potential challenges, and optimize system design, ensuring the project embodies both scientific innovation and practical relevance.

Project Direction Definition

After identifying the overarching goal of "applying synthetic biology to mitigate extreme cold effects," we conducted a comprehensive literature review and prioritized antifreeze proteins (AFPs) as a key research focus. Based on preliminary investigations, we proactively contacted Professor Zhang Lei’s research group from the School of Synthetic Biology and Biomanufacturing, initiating a foundational dialogue.

Professor Zhang Lei highlighted that AFPs exhibit key characteristics such as thermal hysteresis activity and high ice recrystallization inhibition (IRI) activity, making them ideal biomolecules for low-temperature applications. The laboratory typically employs expression vectors such as pET28a for recombinant AFP production. Furthermore, given the variability in activity and stability among AFPs from different sources, we selected 11 family-representative AFPs, establishing a clear molecular basis for subsequent experimental work.

This expert guidance enabled us to swiftly overcome initial technical uncertainties, allowing wet-lab activities to commence efficiently.

Feasibility Assessment

During social exchanges at the Campus Open Day, we identified a significant demand among individuals with sensitive skin for enhanced winter skincare. To further quantify this need and evaluate public perception regarding AFP-fortified hand creams, we conducted a tripartite investigation: a public survey, in-depth user interviews, and technical consultations.

Survey 2.0: Building upon the Society module’s questionnaire, we designed a targeted survey focusing on ingredient preferences, usage scenarios, and performance expectations for antifreeze products. We distributed and collected 112 valid responses from university cosmetic clubs. Data revealed that 49.26% of respondents frequently experienced redness, sensitivity, dryness, or peeling during winter, underscoring a substantial market demand for moisturizing and antifreeze cosmetics and reinforcing our decision to integrate AFPs into cosmetic formulations.

Consumer Interviews: Discussions with beauty blogger Anny revealed her personal experience with winter frostbite and regular use of hand creams. She expressed a strong preference for natural, bio-based ingredients over synthetic alternatives due to their eco-friendliness and biodegradability, providing product-side validation for our biomanufacturing pathway.

Extreme User Scenarios: An interview with Professor Yu Ce, a member of China’s 32nd Antarctic Scientific Expedition and professor of College of Intelligence and Computing, highlighted severe challenges such as frequent frostbite and peeling during field operations, which conventional products failed to address. This confirmed the urgent need for high-performance bio-antifreeze agents in extreme environments.

Biomanufacturing Consultation: We consulted Professor Li Bingzhi, who specializes in lignin conversion and high-value bioproduct synthesis. He affirmed that employing metabolic engineering strategies to develop microbial chassis for antifreeze component production offers significant scalability potential and aligns with the cosmetics industry's growing emphasis on sustainable ingredients.

These collective insights confirmed our strategic focus on the fashion and cosmetics track.

Feasibility Screening: Rational Abandonment of Alternative Directions

After finalizing our primary track, we considered applying AFPs to prevent freezing in concealed door handles widely used in new energy vehicles—a design prized for its aesthetics and aerodynamic benefits but prone to ice blockage in winter. However, through discussions with industry sales specialist Wu Shangmeng and policy research, we learned that mandatory extreme cold testing for vehicles has been enforced in China since 2024, and low-cost physical antifreeze solutions are already commercially available. Persisting in this direction would have misallocated resources relative to market needs. Notably, subsequent regulatory proposals to restrict concealed handle designs further validated our decision, demonstrating the importance of adaptive risk management in synthetic biology projects.

Strategic Input from iGEM Ambassadors

To refine our project strategy, we consulted with two distinguished iGEM Ambassadors, who provided crucial guidance from technical and application perspectives.

Dr. Cai Yizhi, an iGEM Ambassador and seasoned judge, advised on our protein engineering approach. He recommended using metagenomic sequencing of Arctic samples to discover novel antifreeze proteins as ideal templates for rational design. This insight directly influenced our shift to a structure-based engineering strategy. Additionally, he highlighted the importance of Human Practices, urging us to assess public acceptance through surveys, which ensured our project's ethical alignment.

Mr. John Cumbers, another iGEM Ambassador and renowned leader in synthetic biology, strongly validated our product direction. He endorsed the development of topical skincare products utilizing AFPs as both highly feasible and impactful, reinforcing our commitment to the cosmetics track and boosting our confidence in its real-world applicability.

Dialogue with these iGEM Ambassadors provided invaluable strategic perspective, bridging our technical work with ethical considerations and translational potential.

Expert Guidance & Project Development

After selecting cosmetics as the primary application direction, we actively sought expertise from both internal and external authorities. We utilized online resources for data collection, cross-referenced information with faculty and their research teams, and engaged directly in experimental design. Regular progress reviews and effective task delegation accelerated the experimental timeline.

Under the guidance of Professor Zhang Xiangyu, we performed several conventional downstream characterization assays, including:

Antifreeze System Optimization

Bloomage Biotech: Transition from Single Protein to Composite System

Experts from Bloomage Biotech advised that single-component antifreeze agents often underperform in practical settings and that AFPs alone may be insufficient for complex real-world conditions. They recommended adopting a composite antifreeze strategy. Accordingly, we expanded our design scope beyond single-AFP expression to explore multi-component synergistic antifreeze pathways.

Introduction of Trehalose System and Surface Display Technology

Guided by industrial feedback, we began investigating multi-gene circuits. Through collaboration with Professor Wu Yi, we learned that gene duplication on yeast chromosome 3 can significantly enhance stress resistance. Consulting with researcher Xu Jiayu, we identified a natural compatible metabolite—commonly used in cosmetics for its dual antifreeze and moisturizing functions—from chromosome 3-associated compounds and incorporated it into the engineered strain’s synthetic pathway.

Additionally, following recommendations on surface display technology, we localized AFPs on the cell surface to enhance ice-binding efficiency and improve overall antifreeze activity.

Experimental Design & AFP Performance Evaluation

In the initial project phase, we designed a series of point mutants using ESM-1v (Evolutionary Scale Modeling) predictions. However, wet-lab results indicated that these computationally high-scoring mutants failed to deliver expected performance improvements. This outcome prompted critical reflection.

Through discussions with experts PangXinyang and YueXiaoyan , we recognized that traditional site-directed mutagenesis often requires lengthy iterative cycles with limited functional scope. We therefore shifted our protein engineering strategy from sequence-based "forward design" to structure-centric "inverse folding" design.

Using ProteinMPNN as our core design tool, we generated numerous physically stable "non-natural" protein variants with predefined functional conformations, significantly increasing the likelihood of obtaining high-performance AFPs.

As the project advanced, we observed that although some mutants scored highly in ESM and ProteinMPNN models, wet-lab performance remained suboptimal. Consultations with Dr. Yang from BGI highlighted the need for a multi-dimensional evaluation system to improve prediction reliability. We subsequently developed an integrated computational screening pipeline incorporating structural prediction (AlphaFold2), free energy evaluation (FoldX), solubility assessment (Protein-sol), and machine learning-based activity prediction. This pipeline enhanced virtual screening accuracy and provided more reliable candidate sequences for wet-lab validation, reducing R&D costs and timelines.

Molecular Dynamics Simulations

Predictive tools such as AfpPropred and target-freeze primarily assess primary structure, overlooking tertiary configurations. Professor Dong Min recommended molecular dynamics simulations to probe stability and flexibility.

However, these intrinsic properties did not directly correlate with ice inhibition performance. Subsequent discussions with Professor Zhang Lei introduced the F4 program, which quantifies ice-water content in mixed systems, enabling direct correlation between protein structure and antifreeze activity for precise functional characterization.

This integrated approach completes a comprehensive dry-lab platform supporting full-cycle protein design, prediction, and simulation.

In the process of project promotion, the industrial module plays a role similar to the "Golgi apparatus" in processing, integration, and transformation, systematically modifying and maturing the original technology in the laboratory stage to make it ready for market promotion. We continuously optimize the commercialization form and industrialization path of technology products through multi-level interaction with enterprises and consumers. This module revolves around four stages: "Exploring Industrialization Paths," "Benchmarking anainst the Industries," "Insight into Consumer Terminals," and "Integrating Industrial Intelligence." It gradually completes the transformation from inexpert technical concepts to mature commercial products, ultimately promoting projects to enter the market in a complete and applicable form, and achieving effective value release.

The exchange with the SNEFE brand team of Jiangxi Chuhua Cosmetics Co., Ltd. is the "first lesson" for our project to meet industry demands. This dialogue not only examines our technological ideas under industrial standards, but also lays a practical foundation for our future technological roadmap and product positioning.

1. Identifying technical bottlenecks: Transdermal absorption and molecular weight challenges

After listening to our explanation and introduction of the project, as well as the innovative points, Engineer Qiu from SNEFE Brand Research Institute pointed out that the molecular weight of proteins is the key to determining whether they can effectively penetrate into the skin and achieve efficacy. At present, the molecular weight of antifreeze proteins is about 26000 daltons, above the ideal transdermal absorption threshold, which limits their actual bioavailability in cosmetics. To this end, the experimental team began exploring the conversion of large molecule antifreeze proteins into short peptides. The specific improvement is reflected in the de novo design and short peptide generation to enhance its skin penetration ability while maintaining antifreeze activity.

2. Pay attention to safety and stability

Allergy is an important area of concern for cosmetics, and Engineer Qiu further emphasized that protein components have a high risk of sensitization. The team therefore incorporated allergy testing and microbial control into the core process of product development, and referred to the "Catalogue of Used Cosmetic Ingredients" for compliance comparison to ensure the safety and stability of the product in real use scenarios. Meanwhile, we also conducted a comprehensive evaluation of our protein using different allergen prediction models.

3. Strengthen cost and process awareness

After the discussion, we visited the production workshop and product inspection equipment of SNEFE Research Institute. The industry pointed out that the purity and performance indicators that are of concern in the laboratory need to give way to cost control in actual production, including raw material costs and time costs.

This feedback prompted us to choose a surface display chassis in subsequent experiments. We considered multiple factors, and after the bacterial surface displays antifreeze proteins, we can wash the bacterial cells by centrifugation and resuspension, then crush them, and add these crushed cell walls to cosmetics to achieve the same effect. We don't need to purify proteins in complex culture media, which can greatly save costs.Furthermore, considering the public's requirements for the scent of skincare products, we have chosen to use saccharomyces cerevisiae (brewer's yeast) instead of engineered strains to avoid the impact caused by odors.

This exchange also reflects the linkage between "research" and "thinking" in the HP model. We not only listen to feedback, but also actively reflect on the rationality and market adaptability of the technological path, ensuring that the project has industrialization genes in the early stages.

In the in-depth communication with leading biotechnology company Bloomage Biotechnology Corporation Limited, the team has elevated their perspective from "productization" to "industrialization" and "value orientation". This meeting focuses on the application prospects and product competitiveness of antifreeze proteins in the beauty industry, and involves key industry pain points and safety assessments.

1. Driving Technology Solution Iteration: From Single Mechanism to Collaborative System.

The Bloomage Biotechnology Corporation Limited team pointed out that if the application of antifreeze proteins in cosmetics relies solely on a single mechanism, their effectiveness and market competitiveness may be limited. This feedback prompted us to quickly adjust our experimental direction, expanding from focusing on the single function of antifreeze proteins to constructing a "trehalose antifreeze protein" synergistic antifreeze system. This system efficiently synthesizes trehalose inside engineering yeast cells and displays antifreeze proteins on the cell surface, forming a dual low-temperature protection mechanism that effectively inhibits ice crystal growth and reduces ice crystal size. The team has chosen trehalose as the industry recognized moisturizing gold formula, which can also maintain cell barriers and protect biomolecules.

2. Industry-Oriented Safety and Cost Control: The Huaxi team emphasized that products must strictly comply with safety standards and cost feasibility. Accordingly, we integrated allergenicity testing and production safety into essential procedures, while also optimizing expression and purification strategies early in R&D to balance compliance, cost efficiency, and scalability potential.

We learned through research that antifreeze proteins have been used in the food industry for a long time. Therefore, we delved into the food industry and participated in ice cream tasting exhibitions, shifting our perspective from the B2B industry to the B2C consumer end, facing end users directly, and verifying the acceptance and applicability of products in real scenarios.

Understand the specific process from production to implementation: Compared to cosmetics, food has stricter safety standards and regulatory requirements. The public has natural concerns about the allergenicity of "protein" additives. Through in-depth communication with enterprises, we have realized that antifreeze proteins must follow a four stage development path from laboratory to industrialization: firstly, reverse design the research and development route with the goal of food grade safety and cost control, optimize the expression and purification process; Secondly, it is necessary to complete the compliance certification of food additives and conduct toxicology and allergenicity evaluations; Subsequently, establish a food GMP production line to achieve stable and compliant large-scale manufacturing; Ultimately, promote product implementation through food ingredient declaration and market education.

All industry insights ultimately converge and are reflected in the continuous iteration of our business plan. Every conversation with the industry directly translates into more profound and persuasive content in the proposal.

Business Plans

1. Technical solution section: The optimized molecular weight design, purification process, and safety testing process based on industry feedback have become the most prominent differentiation advantages and risk response strategies in the technical chapter.

2. Market and Competition Analysis: Based on our understanding of the industry landscape and stakeholders, our analysis is no longer just general, but has profound industry insights and clear competitive positioning. For example, in STP analysis, we clearly identify "low-temperature workers" and "winter sports enthusiasts" as the core groups.

3. Production and cost planning: The cost awareness obtained from the enterprise makes our production estimates and financial forecasts more realistic, avoiding the idealization tendency commonly seen in student projects.

4. Risk control module: The first-hand risk points learned from the industry, such as security, regulations, and public acceptance, are listed as special topics for detailed discussion, and specific response paths are proposed, greatly enhancing the maturity and credibility of the business plan.

Based on our communication and insights with cosmetics companies, we have pushed the antifreeze protein technology to the productization stage. This independently developed hand cream sample is not only a proof of laboratory concept, but also a crucial step in exploring the commercialization path of antifreeze proteins in the personal care field.

1.Cosmetics substrates with different formulas:

2.Product Display:

The industrial module, like the "Golgi apparatus" in the secretion protein model, has the ability to be released to the extracellular market after processing and modifying project content and technical technology. Our continuous "research" and "thinking" (mitochondria) empower the entire process, ensuring the continuous optimization of the project at the technical, market, and strategic levels.

In the end, the integration of all this industrial intelligence has enabled our antifreeze protein project to no longer be satisfied with experimental results, but to grow into a truly rooted solution with the potential to change the industry.

To explore ways to translate the outcomes of our experiment into practical applications, we took the initiative to step out of the laboratory and engage in dialogues with staff from relevant government departments and professors from law schools.

From interviews with staff at government heating management departments, we learned that current mainstream antifreeze measures for heating in China still rely heavily on industrial salts and ethylene glycol-based chemical antifreeze agents. According to the “2023 Statistical Yearbook on Urban and Rural Construction”, the total centralized heating area nationwide reached 14.324 billion m² by the end of 2023. Based on typical industry parameters, the estimated usage of ethylene glycol during a single heating cycle is approximately 43,000 tons.

However, ethylene glycol is a toxic compound. If ingested in large quantities by humans, its metabolites can cause kidney crystallization and kidney function damage. Furthermore, if such substances leak into the environment through pipelines, they can lead to soil compaction and water pollution, posing significant ecological risks. Chloride-based antifreeze agents also present severe problems.

At the same time, we compare the conditions of different countries. The U.S. Environmental Protection Agency (EPA) stated in its ’Environmental Risk Assessment Report on Winter Road Deicing Agents’ that chloride-based snow-melting agents are widely used due to their low cost and high ice-melting efficiency. According to the report, the cost of environmental corrosion caused by chlorides in the U.S. can account for 4% of the Gross National Product (GNP). Over 100,000 bridges nationwide suffer from structural damage, with repair costs ranging from 78 billion to 112 billion U.S. dollars. A survey conducted in Copenhagen, Denmark, revealed that 50% of 102 bridges exhibited severe steel bar corrosion, with the use of chloride-based snow-melting agents identified as the primary cause.

In contrast, antifreeze proteins produced via synthetic biology technology offer advantages such as “biodegradability” and “excellent environmental compatibility”, without causing long-term pollution. Although the current production cost of biological antifreeze proteins remains relatively high, they already demonstrate certain economic viability and large-scale production potential compared to naturally extracted products. As technology matures and production processes are optimized, their costs are expected to decrease further. In the long run, the use of biological antifreeze proteins will not only reduce environmental governance expenditures but also deliver favorable comprehensive economic benefits.

We firmly believe that while continuously innovating and iterating experimental products, we must also uphold respect for legal regulations. When we approached Tianjin University Law School of Tianjin University with our preliminary conceptualization of antifreeze protein technology, we sought more than just a compliance check—we aimed to find a solid social foundation for our project. Unexpectedly, this dialogue became a core driver for the comprehensive upgrading of our project.

In-depth exchanges with Professor Mi Wei made us realize that the commercialization of synthetic biology products is a journey built on three pillars: “safety”, “evidence”, and “compliance”. We clarified the positioning of antifreeze proteins as "new cosmetic ingredients," and their filing process must be centered on full-chain, traceable safety data. Appealing functional claims such as "repair" and "soothing" are contingent on meeting rigid evidence requirements, including human efficacy evaluation tests. Professor pointed out that regulators are establishing a refined assessment framework for synthetic biology-derived ingredients, covering aspects such as strain traceability and the complexity of metabolic products at the source.

During the conversation, we recognized that an excellent business plan must not stop at describing market prospects—it must prioritize "regulatory compliance." Based on our discussions with the professor, we conducted a "compliance restructuring" of our business plan:

• We transformed the vague category of "policy and regulatory risks" into specific, assessable, and plannable issues, such as "uncertainty in the filing cycle for new ingredients," "failure to obtain evidence for efficacy claims," and "environmental leakage of genetically engineered strains."
• In the SWOT analysis, we added "establishing full-chain safety evidence" as a core Strength (S), and identified "responding to rapidly changing regulatory policies" as a Weakness (W) that must be addressed. Corresponding to this Weakness, the Opportunity-Weakness (OW) strategy explicitly outlined a concrete plan to "engage regulatory expert consultants."

Legal guidance ultimately fed back into the most fundamental aspect: experimental design. To address the "strain traceability" and "biosafety" requirements emphasized by Professor Mi, we implemented two key innovations in our wet experiments:

1. Systematic Strain Traceability and Archive Establishment: We no longer viewed the EBY100 yeast strain merely as a tool, but instead established a comprehensive "identity file" for it. This file details the strain’s origin, genetic background, and all gene-editing operations, laying a solid foundation for the "strain information traceability" required in future new ingredient applications.( For more details, please refer to the safety page.)
   

   Figure 53. Systematic Strain Traceability of EBY 100
2. Biosafety Experimental Testing: To proactively assess and mitigate potential risks, we took the initiative to design a "biocompatibility experimental verification" process. This preliminary safety test is not only intended to meet compliance requirements but also to identify and control biosafety risks in the early stages of product development, providing preliminary data support for subsequent toxicological evaluations.

These regulatory requirements have driven us to establish a full-chain safety assessment system, covering strain traceability, impurity control, and sensitization testing. This ensures that our technology does not cross legal boundaries when it is commercialized.

Just like the secretion process of proteins, the Government module is not an end point, but a starting point for releasing the project’s value to society.

Through dialogues with government authorities and legal experts, we have gained a profound insight: the success of a synthetic biology project depends not only on technological breakthroughs but also on understanding policies and arranging compliance measures. We will continue to advance the project through four-dimensional collaboration—research, education, industry, and government—to enable antifreeze proteins to play a role in broader fields and fulfill our mission of "technology for good."