Background: Soybean Root Rot

Soybean as an Economically Important Crop

Soybeans are among the most economically important food and oil crops globally, valued for their high content of protein, oil, vitamins, and essential minerals. Their adaptability to diverse environments supports widespread cultivation for human consumption, animal feed, and biodiesel production. In China, adjustments in agricultural supply-side structure have led to a steady annual expansion of soybean planting area. Notably, Heilongjiang Province—situated within one of the world’s three major black soil zones—offers highly fertile soils, favorable climate conditions, and a premium ecological environment, making it a core region for soybean cultivation. This province accounts for nearly half of China’s total soybean planting area, with continued area expansion observed in recent years.

Soybean Yield Reduction due to Root Rot Disease

However, the vital role of soybeans is in stark contrast to the devastating losses inflicted by soybean root rot, a disease that severely compromises global yields. Soybean root rot, resulting from complex infections by multiple pathogens such as Fusarium oxysporum, has emerged as a major constraint to the global soybean industry. In the U.S. Midwest soybean belt, annual losses due to this disease are estimated to exceed $1 billion. Similarly, in China’s Northeast Black Soil Region, severe field yield reductions can surpass 30%, while global annual yield losses are estimated to range between 10% and 40%, with extreme cases leading to total crop failure. This widespread damage underscores the severe threat posed by soybean root rot to the sustainable production of soybeans worldwide [1].

In China, the area affected by soybean root rot is projected to reach approximately 667,000 hectares by 2025. The incidence is extremely severe in Heilongjiang (one of the main producing areas of soybean in China), with a particularly high incidence in localised waterlogged areas where farmers often practice continuous monocropping. Root rot is strongly correlated with continuous monocropping, which farmers repeatedly plant soybeans in the same field for convenience and short-term gain.

Research clearly shows that the root rot disease incidence in fields that have been continuously cropped with soybeans for over three years is 2-3 times higher than in fields implementing rotation with crops like corn or wheat. Long-term continuous cropping leads to the accumulation of root rot pathogens in the soil, while metabolites secreted by plant roots create favorable conditions for pathogen proliferation. For example, root exudates from soybeans—such as L-arabitol, amino acids, and organic acids including citric acid and malonic acid—significantly promote chemotaxis, spore germination, and rhizosphere colonization by pathogens like Fusarium oxysporum. This gradually degrades the soil environment and substantially increases the risk of disease occurrence [2].

Methods for Controlling Root Rot Disease

Chemical intervention such as pesticides application can temporarily relieve the disease situation, but long-term use of chemicals faces dual challenges: The frequent use of single-site-of-action fungicides such as metalaxyl (a phenylamide) often leads to the development of resistance in pathogen populations [3]. Furthermore, the repeated application of metalaxyl can negatively affect the structure of soil microbial communities, for instance, by inhibiting beneficial nitrogen-fixing bacteria like Rhizobium and Mesorhizobium, thereby disrupting soil ecological balance [4]. As illustrated by published statistical figures, the application of pesticides in Heilongjiang leads to >60% resistance to common agents and a 25% reduction in beneficial soil Actinobacteria, alongside an 18% increase in potential pathogens, exacerbating ecological imbalance and water pollution.

Due to the widespread occurrence of soybean root rot in China, the exacerbation of this disease through continuous cropping and the ecological issues associated with chemical control, the use of biological control methods using beneficial organisms has become essential for overcoming the challenges of root-rot disease management. These approaches rely on beneficial microorganisms directly inhibiting pathogens (e.g. through antagonism or competition for niches) or activating the soybean's own defence system via immune elicitors. This offers a more environmentally friendly way to reduce the threat of disease.

What We Plan to Do

To enhance the precision and efficacy of biological control methods, we plan to use synthetic biology techniques to engineer functional microorganisms and bioactive molecules in order to address root rot challenges systematically, thereby providing a scientific foundation for transitioning from chemical-dependent controls to ecologically integrated, sustainable solutions that are equally effective or even more effective in long term.

Stakeholder Engagement

Guided by a reverse logic framework centered on stakeholder engagement (from end-user needs), we carried out our human practices research. We conducted in-depth interviews with key stakeholders across the soybean agricultural chain to identify their specific pain points, technical needs and application scenarios. This ensures that our project closely align with real-world challenges, we conducted structured engagements across five key dimensions:

Product/User End: Held focused dialogues with farmers facing production challenges, to capture their practical needs regarding control efficacy, ease of use, and cost-effectiveness of biocontrol agents.

Technical End: Held discussions with experts researching in agriculture fields to obtain professional insights on root-rot disease mechanisms, strain-engineering techniques, implementation of biological control methods and recommended future optimization strategies.

Production End: Collaborated with synthetic biology companies to understand their technical preferences, challenges in strain fermentation (soil-microbial agent production), scale-up process from bench and pilot scale to industrial production and regulatory constraints in microbial agent production.

Product Form Exploration: Engaged in targeted discussions with synthetic biology enterprises within the agricultural filed, confirming that powdered inoculants represent the most suitable formulation for green agricultural applications.

Education End: Conducted in-depth conversations with farmers and students, capturing their practical needs for interactive and interesting synthetic biology education carriers, the adaptability of knowledge dissemination forms to students of different cognitive levels, and the integration points of popular science content with campus curriculum systems.

Guided by frontline feedback and expert advice, the direction of our project's R&D evolved iteratively–transitioning from an initial goal of enhancing bacterial antagonism to the development of an advanced powdered inoculant. This mixed microbial agent is designed to become active in the soil (the powdered microbial agent revives from spores into its active vegetative state), where it delivers chitinase and β-amyrin. The resulting product effectively addresses the need of an cost-effective, efficient, user-friendly solution for farmers while fulfilling the scalability and regulatory requirements of industrial production. It represents an economically viable and environmentally sound strategy for combating soybean root rot and promoting sustainable agricultural practice.

We firmly believe that agricultural research must be anchored in core human needs, such as food security, ecological protection and the requirements of farmers, in order to truly validate its value and long-term impact. We draw inspiration from real-world agricultural scenarios and continuously enhance our capabilities through global knowledge and practical experience. Our goal is to return more efficient, safe and economical agricultural technologies to the fields, safeguarding crop health and food security through scientific research. We strive to be beneficial contributors to agricultural development and responsible stewards of the ecological environment.

Before embarking on each exploratory journey, we firmly believe that clear, steadfast principles are essential to ensure that our actions stay on course and that our projects advance steadily. These principles provide clear guidance for decision-making and help the team to maintain unified goals and direction amidst challenges. Our Human Practices work consistently follows the PTT (Preparation, Takeaways, To-do List) principle, guiding our actions methodically from conception to practice and from stage goals to breakthroughs.

Our PTT Principle Steps

1. Preparation: To deeply understand our core issue, we proactively build bridges with stakeholders. Through sincere, professional interviews, we listen to diverse voices, providing precise input for project optimization and building a comprehensive cognitive framework.
2. Takeaways: Precise answers to core questions are key to cutting through information clutter and clarifying the current situation. This allows deep insight into the problem's essence and root causes, expands our cognitive dimensions systematically, and lays a solid foundation for subsequent actions.
3. To-do List: This crucial phase not only structures fragmented insights but also charts a clear direction for subsequent actions – pinpointing next core tasks and focus areas. This seamlessly connects and efficiently advances the next Preparation phase, ensuring the project progresses steadily towards its goals.

Product/User End 1: Liantang Farmer Investigation

Liantang Town Farmer Visits

On Liantang's agricultural frontline, we met farmers who were direct participants in, and witnesses to, local agricultural development. Their years of experience with land and crops have given them valuable knowledge of planting and first-hand experience of root rot.

Their farms grow a variety of crops, including large areas of aquatic plants such as water bamboo, rice and lotus root. Some also cultivate dryland maple varieties such as the Japanese Red Maple 'Kohgyoku'. Discussions about root rot were filled with concern and a sense of helplessness.

Preparation

In order to gain an understanding of the farmers' basic knowledge of root rot, its current status, existing control methods and requirements, we designed a set of questions to gather practical information on incidence, field responses and the shortcomings of current solutions. This approach enabled us to develop a comprehensive and realistic understanding of the issue.

1. Identify actual field root rot situations.
2. Understand farmers' existing control methods and challenges.
3. Clarify farmers' needs for control products.
4. Gauge farmers' acceptance and selection habits for new products.

Takeaways

Disease incidence varies: It is lower in aquatic crops (but occurs with prolonged waterlogging) and higher in dryland crops due to soil issues, high temperatures/humidity and herbicides. It has increased recently due to extreme weather. Waterlogging for three days in summer can easily trigger outbreaks. It is diagnosed via leaf and root symptoms. It spreads via infected soil. It can cause yield losses of over 50% or total crop failure. Once symptoms appear, it is basically impossible to cure, making biological prevention the mainstream strategy.

Existing control shortcomings: Farmers use crop rotation and drainage (physical measures which are slow); chemicals are used upon discovery (they are fast-acting, but leave residues and have a poor curative effect if found late). The ultimate solution is prevention. Chemicals cause resistance and residues. Unified control is needed in contiguous plantings. Organic farming uses bio-agents (e.g. Muquan's B. subtilis), but these are ineffective. Control costs currently account for ~20% of expenditure and are rising.

Farmer priorities: Products must be residue-free. An acceptable cost is 20–30 RMB/mu/season. The ideal effect is achieved within 3–7 days of the first symptoms appearing. Preference is given to slow-release granules or foliar sprays.

Acceptance of new agents: We are not opposed to GMO microbial agents, but we require authoritative proof of safety, verification that they are residue-free, and free trials. We learn about new products via news and Douyin (TikTok).

Plan of Action

1. Through interacting with farmers, we gained an understanding of the realities of the field: the incidence varies, and is higher in dryland crops and increasing. The current 'physical + chemical ' methods have multiple pain points (efficacy, residue, cost). Biological method is a better solution for organic agriculture but these are not effective enough. Farmers want safe, quick and affordable products and are open yet cautious about GMO microbes. Prevention is key as a cure is rarely possible.
2. This feedback indicated that understanding user needs was not enough. Solving root rot requires a focus on prevention methods, specifically biological agents.
3. We therefore needed to investigate the real-world efficacy, cost, and usability barriers of mainstream root rot control agents and bio-inoculants on the market.

Product/User End 2: Shanghai Jiading Yuanju Agricultural Technology Service Station

Jiang Xiaoqing has worked at Yuanju Station for over 30 years, evolving from selling traditional pesticides to promoting bio-control products and becoming a pillar of the local community. The station offers chemical agents and bio-inoculants such as B. subtilis, as well as subsidised green agricultural inputs in accordance with the government catalogue. For 30 years, this supply point has adapted to the evolving needs of agriculture, providing reliable products and services and safeguarding bio-control in Jiading.

Preparation

Visiting Yuanju Station aimed to understand promoted bio-control product types, applicable scenarios, root rot control strategies, and compare bio/chemical methods on efficacy, safety, and price. We sought practical agronomic data to provide better farmer advice and optimize bio-control strategies.

1. Primary bio-inoculants used for root rot.
2. Comparison of bio-control vs. chemical pesticide characteristics.
3. Reasons for slow action of bio-inoculants.
4. Mechanism of bio-inoculants antagonizing F. oxysporum.

Takeaways

Inoculants like Bacillus subtilis prevent bacterial diseases and offer some root rot control, recommended for organic agriculture and green farming (some seasonally limited).

Key Differences: Bio-control acts biologically (slow, preventive, safer, complex storage/use). Chemicals act toxically (fast, strong, curative, dosage can be increased against resistance, simpler storage/use usually).

Slow Action Reason: Bio-inoculants rely on beneficial microbes colonizing, reproducing, and establishing dominant populations in the rhizosphere. This adaptation and population build-up to effective thresholds (for competition, antimicrobial production) takes time, unlike direct chemical action.

This survey clarified bio-control product types/uses promoted by ag-tech stations, differences from chemicals, primary root rot prevention methods, and the prevalent issue of slow action.

To-do List

Literature Review: B. subtilis significantly controls root rot by colonising roots, competing with pathogens and inducing plant resistance (e.g. by activating antioxidant/defence pathways). It also secretes cell wall-degrading enzymes (e.g. chitinase and cellulase), as well as antagonistic compounds, siderophores and hormones that directly inhibit pathogens [5]. Furthermore, studies on other microbes show that a significant reduction in chitinase secretion leads to reduced pathogen inhibition [6]. This highlights chitinase as a key functional factor in B. subtilis-mediated root rot control. However, natural chitinase secretion is often limited by regulatory mechanisms and is therefore insufficient for efficient field control. Therefore, we plan to genetically engineer bacterial strains to enhance the expression of the chitinase synthesis gene, with the aim of creating engineered strains with stronger and more stable antagonism to improve bio-control resources.

Define Project Direction: Focus on 'engineering bacterial strains to enhance chitinase production'. Upstream design (strain genetic modification and chitinase expression regulation) is critical and requires priority attention due to the many unresolved technical challenges it contains. To expedite progress, we have chosen E. coli as the initial research system for chassis development.

Considering the need to build E. coli expression systems for chitinase, we planned expert interviews focusing on upstream design, common technical bottlenecks, and preventive strategies for traditional biocontrol agents targeting Fusarium. This includes experimental design validation and optimization of key functions, such as enzyme secretion efficiency and antagonistic activity.

Technical End 1: Expert Interviews with Deputy Researcher Wang Jinbin, Shanghai Academy of Agricultural Sciences, Biotechnology Institute

Deputy Researcher Wang Jinbin has extensive experience in agricultural biotechnology, specializing in "enzyme expression technology" to address agricultural environmental and food safety issues. His research approach highly aligns with our team's direction.

Preparation

To advance upstream experimental design using E. coli, we formulated questions on "chitinase expression strategies in E. coli":

1. Key optimization factors for constructing functional enzyme (e.g., chitinase) expression systems in E. coli?
2. How does fermentation process directly link to product form and field efficacy? Practical solutions for functional molecule adsorption/degradation in soil?

Takeaways

1. Gene Sequence Optimization is paramount for expressing functional enzymes like chitinase in E. coli. Must adjust codons based on E. coli preference (replace rare codones, avoid stacking) to reduce ribosomal stalling and ensure translation efficiency/protein yield.
2. Given E. coli's limited native secretion capacity, adding/optimizing secretion signals is core. Must select specific signal peptides highly compatible with the target enzyme and pair with a suitable secretion system to ensure efficient transmembrane transport to the periplasm or extracellular space, achieving secretion goals.

To-do List

Building efficient E. coli chitinase expression systems required a focus on gene optimization, expression element selection and enhancing secretion function to boost expression efficiency and activity. Therefore, we have planned the design of secretory peptides for chitinase. However, the literature revealed over 2,000 reported E. coli secretory peptides, with limited comparative studies on secretion efficiency. As the level of chitinase secretion directly determines the antagonistic function, we have designed dry lab models and plan to use GFP as a model protein to predict the secretion efficiency of different peptides, with the aim of identifying the optimal one for the E. coli system.

Technical End 2: Expert Interviews with Associate Professor Li Yan, Department of Plant Pathology, China Agricultural University

Prof. Li Yan, an experienced expert in agricultural bio-control, plant disease bio-control, and plant-bacteria interactions, leads relevant NSFC and national key R&D projects. Her team received awards (e.g., Shennong China Agricultural Science and Technology Award), possessing solid foundational and applied research experience highly relevant to our project.

Preparation

Considering subsequent commercialization, we prepared questions on technical and application aspects:

1. Advantages/limitations of E. coli as a chitinase expression host?
2. Strategies to improve chitinase activity, stability, and antimicrobial effect?
3. Key considerations for translating iGEM projects into practical applications?
4. Alternative strategies to enhance engineered strain antagonism beyond boosting chitinase?

Takeaways

1. E. coli: Advantages - Fast growth, clear genetics, mature tools, inducible promoters allow high enzyme accumulation, suitable for lab-scale prep, rapid engineering, initial gene testing. Limitations - Lacks eukaryotic protein modification, prone to inclusion bodies, secretion efficiency relies on heterologous signals, weak environmental adaptability.
2. Enhancing Chitinase: Strategies include gene optimization, expression regulation (e.g., inducible promoters like IPTG-induced T7, codon optimization, fusion stability tags to reduce inclusions, improve stability), and formulation adaptation. For soil, use engineered live bacteria for continuous secretion. Different chitinase types have different mechanisms and structures; comparing chitinases from different species could optimize the final effect.
3. Enhancing Antagonism: Consider a "dual-pathway synergy" approach: while expressing chitinase to degrade pathogen cell walls, simultaneously inhibit the pathogen's own chitin synthesis pathway ("degrade + inhibit") for improved efficacy. This could involve finding/expressing a natural small molecule targeting pathogen chitin synthesis.

To-do List

1. Chitinase Selection: Post-discussion with Prof. Li, we gained a more systematic understanding of host selection, enzyme optimization, iGEM translation, and regulations, correcting previous oversimplified views on host choice. We plan to express chitinases from different sources and test their secretion efficiency and enzyme activity.
• [BlJ24] Bacillus licheniformis J24 chitinase (gh18A) - NCBI: MF765958
• [Blchi] Bacillus licheniformis DSM 13 (chitinase) - NCBI: CP000002.3
• [Bschi] Bacillus subtilis chitinase - NCBI: AF069131.1
• [Smchi] Serratia marcescens strain GEI chitinase A - NCBI: GQ855217.1
2. Natural Product Selection: Following Prof. Li's advice, literature review revealed β-amyrin, a natural compound, can specifically target and inhibit pathogen chitin synthase activity, blocking their cell wall synthesis. This complements the "chitinase degradation" strategy, creating a synergistic "degrade + inhibit" mechanism. Based on this, we subsequently constructed engineered strains for heterologous β-amyrin expression, aiming to enhance pathogen control through this dual mode of action.

Academic Exchange: CCiC & Synbiopunk Bio-Developer Conference 2025

The CCiC & Synbiopunk 2025 event gathered 107 iGEM teams from China and 200+ attendees for this synbio and frontier tech integration conference. Our team, led by instructors, exchanged and learned with other high school and undergraduate teams. We also had a special mission: our wet lab work was hindered as the root rot pathogen  Fusarium oxysporum is not on the white list (iGEM safety policy). We sought help from the Chinese iGEM community at CCiC.

The event featured tight scheduling: high-quality academic reports on frontiers, student presentations (posters, talks), and self-organized meetings for flexible discussion on tech applications, project challenges, and implementation, aligning with iGEM HP's emphasis on practical exchange.

Takeaways

1. Participating in CCiC & Synbiopunk 2025 was highly rewarding. Our team stood out among the participants, notably as the only high school team among the award winners, for our outstanding results and collaborative spirit. The event fostered exchange and promoted synbio-tech fusion, clarifying for us students the need to balance innovation with social value.
2. Crucial Collaboration: During exchanges with Tsinghua University's team (Tsinghua-M), we identified their project's S. cerevisiae with smarlet (likely a tag or construct) as potentially suitable for our antagonism assays. After deep communication, they agreed to provide us with this yeast strain. We are immensely grateful for Tsinghua-M's support!
3. Broader Perspective: Discussions highlighted that commercializing projects requires not only technical innovation and lab maturation but also later engagement with agricultural enterprises for fermentation scale-up (lab flask to industrial tank), field validation, and formulation development (e.g., wettable powders for soil application). This insight guided our subsequent HP plans to visit production-end companies.

Production End: Shanghai Bluepha Co., Ltd. – Chen Kang & Xu Guiwen

To precisely define the final product form and ensure technology-market fit, we planned to interview Bluepha, a synbio industry leader, for their expertise in product R&D and industrialization.

As a pioneer, Bluepha, founded in 2016, leads with innovation. Its flagship "Black Light Lab" uses AI/automation to compress traditional strain iteration cycles from 2-3 years to just 6 months, reducing product development time by 70%, breaking R&D efficiency bottlenecks. In agriculture, Bluepha successfully launched bio-inoculants, organic water-soluble fertilizers, etc., aligned with green development needs, improving crop quality, soil health, and reducing chemical inputs, accumulating rich application experience.

Preparation

Previous interviews revealed challenges in bio-control: suboptimal efficacy, lack of targeted products for root rot. We sought Bluepha's insights on industrial production conditions and process optimization to solve existing problems and upgrade the agricultural bio-control industry.

1. What should our final product form be?
2. Key control conditions for industrial strain production?
3. Challenges in strain tech improvement, industrialization, and field application?
4. Advice on strain R&D resource allocation and support?

Takeaways

1. Product Form: Bluepha experts recommended focusing on inoculant formulation, aligning with the chitinase-based root rot control strategy. Inoculants allow continuous chitinase production, precisely degrading pathogen cell walls. They avoid chemical pollution, are safer for crops/soil, can colonize soil for long-term suppression, and offer better stability and lower cost compared to single enzyme preps, enhancing project competitiveness.
2. Inoculant Types: Liquid (high water content >80%, prone to stratification, spoilage, contamination, temp/oxygen sensitive, shelf life 6-12 months RT); Powder (can achieve 18-24 months RT shelf life if high quality); Granular (solid, some slow-release, may use binders causing pressure on microbes, internal humidity varies, potential local activity loss).
3. Application: Powder inoculants mix directly with solid fertilizers (chemical/organic), planting substrates (soil, potting mix, coco coir) without pre-dilution. Liquid requires dilution, mixes poorly with solids. Granular may settle unevenly in substrates.
4. Industrial Production: Requires precise control: Temperature is critical (especially during drying liquid into powder, avoid high temps deactivating >80% viable bacteria). Optimize fermentation parameters (materials, O2 concentration). Stabilize environmental temp/humidity to reduce scale-up impact. Control costs ensuring process improvements suit mass production.
5. Challenges: Strain performance often drops during industrial scale-up. Field conditions (high temp, UV) reduce viability and colonization (far below lab levels). Achieving 50% target survival rate may double costs, making it difficult for market acceptance. Current protection tech offers external shielding but doesn't address genetic-level stress resistance defects.

Conclusion: After comprehensive consideration, we prioritized powder inoculant as our target final product form.

To-do List

1. Bluepha experts agreed to let us experience their advanced fermentation and pilot spray-drying equipment. Meanwhile, they clarified the core direction of "taking green agriculture needs as the guide to promote the transformation of engineered bacterial strains into commercial products"—this key takeaway has directly become a crucial starting point for launching the "Product Form Exploration" component of our Human Practices program—marking a solid step toward commercialization!
2. We finalized powder inoculant as the target product form to better align with green agricultural development needs. Discussions also highlighted the importance of biosafety and regulatory compliance, prompting us to consult an experienced lawyer for professional support.

Safety & Regulations: Interview with Lawyer Zhang

To address legal and regulatory aspects of project implementation, we contacted Zhang Zhizhong, Partner at Beijing SanYi Law Firm. He focuses on biotech enterprises, possessing extensive practical experience in technical patent ownership, collaboration rights, and legal solutions for technology transfer, helping companies mitigate risks.

Preparation

Lawyer Zhang accurately grasps legal key points for technology implementation. We inquired about:

1. Approval processes, regulatory standards, and prohibitions for engineered microbes in different regions.
2. Ecological risks of antibiotic resistance genes (ARGs) in engineered microbes and current regulation trends.
3. Liability delimitation towards market and users during commercialization.

Takeaways

Regulations: China: Follows Biosafety Law and Regulations on Administration of Agricultural GMO Safety. Agricultural engineered microbes require 5-stage approval (research, intermediate trial, etc.) reviewed by MARA and biosafety committees. EU: Strict regulation under *Directive 2001/18/EC*, requires comprehensive environmental risk assessment and public consultation; prohibits clinically relevant ARGs. North America:  More product-specific, case-by-case. US: Multi-agency (EPA, USDA, etc.); from 2025, eased scrutiny on non-clinically relevant ARGs. Canada: Focuses on ARG transfer risk; no mandatory public consultation.

ARG Risks & Control: ARGs in engineered microbes risk transfer to other microbes, potentially creating "superbugs" and disrupting soil microbial balance, threatening ecological security. EU: Bans clinically relevant ARGs. China:  Requires safety data (e.g., ARG transfer frequency), encourages marker-free editing. US: Post-2025, non-clinically relevant ARGs not automatically strictly reviewed; focus shifts to plant harm potential. All aim to reduce ecological risk.

Liability: Based on laws (Civil Code, Biosafety Law in China). liable parties bear civil compensation (direct losses, restoration costs), administrative liability (fines, recalls) for crop damage/ecological harm; criminal liability for severe cases. Contracts can define boundaries (e.g., compensation caps, exclusions for misuse). Product labeling with usage guidelines and risk warnings further clarifies responsibility.

To Do Next

This provided crucial understanding of cross-jurisdictional compliance requirements, core risk management, and liability definition for engineered microbes from R&D to commercialization. It deepened our knowledge in international regulatory differences, ecological risk prevention, and the legal boundaries for commercialization, offering legal safeguards from compliance, risk avoidance, and liability clarification perspectives for transitioning from lab tech to market product.

Product Form Exploration: Shanghai Bluepha Co., Ltd. – Sun Qiang & Wu Bingxu

Based on in-depth exchanges with experts from Bluepha, we not only obtained a valuable opportunity to gain experience with their advanced fermentation and pilot-scale spray-drying equipment, but also clarified the core direction of taking green agricultural needs as the guide to promote the transformation of engineered bacterial strains into commercial products—this key insight has directly become an important starting point for us to launch the "Product Form Exploration" component of our Human Practices program.

In light of the practical demands of green agriculture for agricultural inputs, such as easy storage, easy application, low transportation costs, and low environmental loss, we finalized the powdered inoculant as the ultimate target product form after multiple discussions. Subsequently, following the transformation logic of "from engineered bacterial strains to powder products," we carried out phased and systematic exploration of shake flask fermentation, 75-L tank fermentation, and spray-drying processes under the guidance of Bluepha’s experts.

In the early stage of exploring and confirming the product form, shake flask fermentation experiments played a core role in the screening of basic formulations. Through these experiments, we focused on verifying the compatibility of different formulation combinations with the growth status of bacterial strains and spore production efficiency, initially eliminating components that are detrimental to the retention of bacterial strain activity, and screening out potential directions for basic formulations. This step not only defined the focus scope for subsequent scale-up experiments, but also gave us a preliminary understanding of the rules for matching formulations with bacterial strain characteristics, helping us avoid ineffective paths in future explorations.

As the direction of the basic formulation gradually became clear, we proceeded to the 75L tank fermentation experiment phase under the guidance of Bluepha’s experts. The core goal of this phase was to achieve transformation and verification of laboratory formulations for pilot-scale production. Compared with shake flask experiments, 75-L tank fermentation is closer to actual production scenarios. Through this phase, we focused on verifying the stability of the initially screened formulations under scaled-up culture conditions, as well as the growth consistency and production efficiency of bacterial strains in batch culture. This effectively bridged the technical gap between laboratory research and industrial production, and provided more reference-worthy practical data for subsequent process optimization.

After completing the preliminary verification of the fermentation phase, experts from Bluepha, in conjunction with the spray-drying process plan, launched the exploration of transforming liquid fermentation products into powder form. The spray-drying process is a crucial step in achieving the target form of powdered inoculant. This process can convert liquid fermentation products into powder with controllable moisture content and good fluidity—it not only addresses the pain points of liquid formulations (such as short storage periods and high transportation costs), but also fully aligns with the demand for convenient and long-acting inputs in green agriculture. This transformed the technical concept of converting bacterial strains into practically usable products into a preliminary physical form, marking the first solid step toward commercialization.

This preliminary exploration of product form not only clearly established the technical pathway of "shake flask fermentation screening → 75-L tank fermentation scale-up → spray-drying process finalization," but also allowed us to verify, through practice, the high compatibility between powdered inoculants and the needs of green agriculture. The advancement of each phase has been closely aligned with the transformation goal of "from technology to product," laying a solid preliminary foundation for subsequent efforts to further optimize process parameters, ensure biosafety and compliance, and even promote the product’s entry into the market.

Education

Field Education: Rooting Biological Control Knowledge Among Farmers

Synthetic biology is a new concept for farmers, and it is crucial to change their perception of biological control. Therefore, we have carried out popular science education activities, aiming to help more people understand the advantages of biological control as an alternative to chemical pesticides, and learn to use biological control technology to protect crop health and soil ecology.

In the agricultural popular science activities of biological control, we further focused on the core issue of "how to make popular science truly effective" —— Given the large number of farmers in China, as well as the great differences in planting scenarios and knowledge backgrounds, how to make farmers with different needs willing to participate, understand and apply the knowledge was a tough problem we faced initially.

To break through this dilemma, our team took the initiative to design farmer-friendly activities such as on-site field observations and agricultural technology mini-courses: at the planting base, we explained the value of biological control through practical cases including the comparison of control effects and cost accounting; we also produced accessible popular science materials such as graphic manuals and short videos based on farmers’ preferences. These activities not only helped us identify a more precise popular science path and promotion goals, but also made it easier for biological control knowledge to reach the fields. We hope that in the future, teams participating in iGEM and groups around the world concerned about agricultural popular science can draw inspiration from our promotion and exploration, and work together to make the concept of biological control deeply rooted in people’s minds and ensure the effective application of related technologies.

Campus Education: Spreading Synthetic Biology Knowledge and Developing the Bio Uno Game to Enable Students to Learn Synthetic Biology Through Gameplay

For students, synthetic biology is an abstract cutting-edge discipline. It is of great importance to stimulate students’ interest in exploration and help them understand core concepts intuitively. Therefore, we have integrated synthetic biology knowledge into campus activities and developed the Bio Uno game, allowing students to learn synthetic biology knowledge through the game we designed.

In the campus popular science activities, we focused on the core issue of how to materialize abstract knowledge—there are differences in students’ cognitive levels and mastery of biological knowledge, so how to enable students with different foundations to participate actively and understand easily was a challenge we faced in the early stage.

To solve this problem, our team integrated the Bio Uno game into classroom teaching and after-school activities: in class, we guided students to interact in accordance with the game rules—colors corresponded to biomolecules (e.g., red for carbohydrates, blue for proteins) and numbers distinguished the degree of molecular polymerization (e.g., monomers like monosaccharides)", so that students could understand knowledge such as biomolecule classification and polymerization while playing. After class, we organized game competitions and distributed game rule manuals to help students consolidate the knowledge points. These practices not only defined an effective path for campus popular science of synthetic biology, but also made synthetic biology knowledge more accessible to students. We hope that in the future, more campus popular science workers and groups concerned about youth science education can gain insights from our practice, and work together to promote the popularization of synthetic biology knowledge on campus and ignite students’ passion for scientific exploration.

Summary

The research followed a "reverse logic from end-user needs" framework and the PTT (Preparation, Takeaways, To-do List) principle, interviewing multiple stakeholders:

Product/User End

Our Human Practices research on the biological control of soybean root rot was driven by the following factors: this disease causes a 15-50% global yield loss, with an estimated 10 million mu (a Chinese agricultural land unit) of affected areas in China by 2025; its incidence is exacerbated by continuous cropping; and chemical control methods pose ecological problems—all of which make biological control a key development direction.

Farmers in Liantang pointed out that biological control is a more suitable solution for organic agriculture, but existing biological products are not effective enough. This led us to focus on the value and existing issues of microbial inoculants in addressing soybean root rot.

Jiang Xiaoqing from Yuanju Station clarified that chemical agents take effect quickly but have drawbacks, while biological inoculants are safe but slow-acting. This prompted us to define our project direction: enhancing the chitinase production capacity of microbial inoculants to improve their antagonistic function.

Technical End

Prof. Wang Jinbin confirmed that the expression of chitinase requires the optimization of secretion signals. Thus, we developed an algorithm capable of predicting the secretion efficiency of different secretory peptides. We tested 2000 secretory peptides and selected the one with the strongest secretory capacity in model organisms. Additionally, we tested the secretion efficiency of chitinases from different genera using this optimal signal peptide. Additionally, we tested the secretion efficiency of chitinases from different genera using this optimal signal peptide.

Prof. Li Yan proposed the synergistic strategy of "chitinase degradation + β-amyrin inhibition," which led to the selection of 4 chitinase genes and β-amyrin. Following Professor Li’s advice, a literature review revealed that β-amyrin—a natural compound—can specifically target and inhibit the chitin synthase activity of pathogens, blocking their cell wall synthesis. This complements the "chitinase degradation" strategy, forming a synergistic "degrade + inhibit" mechanism. Based on this, we subsequently constructed engineered strains for the heterologous expression of β-amyrin, aiming to enhance pathogen control through this dual mode of action.

Academic Exchange: At the CCiC & Synbiopunk 2025 event, we established a crucial collaboration with Tsinghua-M team, focusing on Saccharomyces cerevisiae, which greatly facilitated the conduct of antagonism experiments.

Production End: Bluepha consultation confirmed powder inoculant as the final product form, as this better aligns with the development needs of green agriculture.

Safety/Regulatory End: Lawyer Zhang clarified international regulatory differences, ARG risks, and commercialization liability issues.

Product Form Exploration: This preliminary exploration of product form not only clearly established the technical pathway of "shake flask fermentation screening → 75-L tank fermentation scale-up → spray-drying process finalization," but also allowed us to verify, through practice, the high compatibility between powdered inoculants and the needs of green agriculture.

Education End: These activities not only helped us identify more precise popular science paths and promotion goals but also made biological control knowledge more accessible to rural areas. Meanwhile, these practices not only defined effective paths for campus popular science but also made synthetic biology knowledge more easily acceptable to students.

Based on this feedback, our R&D pivoted from simply enhancing B. subtilis antagonism to designing a mixed agent expressing chitinase and β-amyrin. This aims to promote a shift in disease control from "chemical dependency" towards "ecological synergy", contributing to the security of the soybean industry.

References