For us, the core of Human Practices (HP) lies in transforming understanding of a problem into actionable implementation. We begin with investigation and listening, but measure our effectiveness through design decisions, biosafety measures, and implementation pathways.

Our integrated Human Practices (iHP) focuses on how different stakeholders — patients and families, clinicians, microbiome and synthetic biology experts, traditional Chinese medicine and natural product researchers, regulatory and industry partners, and the public — perceive colorectal cancer (CRC), tangerine peel / naringin, and living probiotic therapeutics. By thoroughly understanding these perspectives, we refine what to do, how to do it safely, and how to truly deliver the outcome to patients.

To ensure comprehensiveness and practicality, we adopt two complementary pathways:

• Needs and Evidence Pathway — using surveys and interviews to identify pain points (such as the trend of early-onset CRC and treatment burden), assess public awareness of tangerine peel / naringin, and validate the vision and feasibility of our “EcN + targeting + natural product” approach;
• Implementation and Safety Pathway — co-designing with stakeholders a biosafety system (dual suicide switches), clinical and regulatory translation plans, manufacturing and accessibility strategies (e.g., freeze-dried formulation), and responsible science communication.

Through this dual-pathway framework, our insights from research are directly translated into technical design, risk management, and implementation proposals, forming a closed loop from problem discovery to real-world impact.

We ultimately identified three dimensions most aligned with the iGEM project: Safety, Accessibility, and Impact.These three pillars ensure that our project is not only scientifically feasible but also responsible in ethical, social, and practical terms.

• Safety — Ensuring the safe use of the engineered probiotic through a dual kill-switch system design, regulatory compliance review, and ecological risk minimization.
• Accessibility — Focusing on cost control, user convenience, and adaptability for diverse patient groups.
• Impact — Evaluating the solution’s potential to alleviate colorectal cancer burden, advance scientific progress, and create long-term value for patients and healthcare systems.

Guided by these three dimensions, our responsibility framework provides a structured foundationfor project design decisions, stakeholder communication, and feasibility assessment.

When constructing our responsibility framework, we aimed to balance medical value, biosafety, and social acceptance.

Ultimately, we defined three core dimensions that best align with the iGEM philosophy: Safety, Accessibility, and Impact.

These three pillars allow us to evaluate whether our project is not only scientifically feasible, but also ethical, socially responsible, and applicable in real-world contexts.

• Safety — Ensuring the safe use of engineered probiotics through the dual suicide system, regulatory compliance reviews, and minimization of ecological risks.
• Accessibility — Emphasizing cost control, ease of use, and adaptability across different patient groups.
• Impact — Assessing our project’s contribution to reducing the burden of colorectal cancer, advancing scientific progress, and creating long-term value for both patients and the healthcare system.

Guided by these three dimensions, our responsibility framework provides a structured foundation for design decisions, stakeholder communication, and feasibility evaluation, ensuring that responsibility is embedded throughout every stage of our project.

Stakeholder	Role and Contribution
Traditional Chinese Medicine (TCM) Experts	Provided insights into the traditional functions and pharmacological understanding of tangerine peel (Chenpi).
Scientific Researchers	Clarified the scientific basis underlying the bioactivity and efficacy of tangerine peel compounds.
Oncologists / Gastroenterologists	Explained the rising trend of early-onset colorectal cancer (CRC) and the clinical treatment bottlenecks faced in practice.
Cancer Epidemiologists	Offered population-level data and evidence-based analysis to support project relevance.
Early-onset CRC Patients and Families	Shared firsthand experiences, needs, and pain points, helping shape user-centered design.
Health Media / Science Communicators	Helped identify public perception gaps and enhanced science communication effectiveness.

Stakeholder	Role and Contribution
Traditional Chinese Medicine (TCM) Experts	Provided insights into the traditional functions and pharmacological understanding of tangerine peel (Chenpi).
Scientific Researchers	Clarified the scientific basis underlying the bioactivity and efficacy of tangerine peel compounds.
Oncologists / Gastroenterologists	Explained the rising trend of early-onset colorectal cancer (CRC) and the clinical treatment bottlenecks faced in practice.
Cancer Epidemiologists	Offered population-level data and evidence-based analysis to support project relevance.
Early-onset CRC Patients and Families	Shared firsthand experiences, needs, and pain points, helping shape user-centered design.
Health Media / Science Communicators	Helped identify public perception gaps and enhanced science communication effectiveness.

Since April, we have gradually established an integrated Human Practices (iHP) cycle, ensuring that our project continuously evolves through ongoing stakeholder engagement, feedback absorption, and technical optimization.

This dynamic cycle enables our design to better address social, medical, and ethical needs.

Renew

The first step of our iHP cycle is Renew, or updating our understanding.

By engaging with diverse stakeholders, we constantly refreshed our knowledge of early-onset colorectal cancer (CRC), re-examined our initial assumptions, and ensured that our project remained aligned with real-world challenges.

For example, through conversations with oncologists and epidemiologists, we learned that early-onset CRC is rapidly increasing in incidence, with severe side effects and high treatment costs.

This insight clarified our direction — our project must not only achieve scientific innovation, but also address patients’ real needs, by developing a new approach that integrates engineered probiotics, natural products, and targeted delivery.

Record

The second step is Record.

We systematically collected and analyzed stakeholder feedback, tracking how these perspectives shaped our experimental design, product concept, and implementation pathway.

Examples include:

• Discussions with TCM and natural product experts confirmed that naringin is the key bioactive compound responsible for Chenpi’s anti-inflammatory and anticancer effects, solidifying our plan to construct its synthetic pathway.
• Conversations with patients and families revealed their concerns about treatment costs and daily convenience, which inspired us to evaluate whether a freeze-dried bacterial powder or oral formulation would be more accessible.

Reflect

The third step, Reflect, focuses on evaluating and adjusting the project based on collected feedback, ensuring scientific rigor, ethical responsibility, and sustainability.

For instance:

• In response to doctors’ safety concerns, we incorporated a dual suicide system (pBAD + pCspA → MazF) to strengthen both environmental and user safety.
• Input from investors and intellectual property specialists prompted us to explore cost reduction strategies and patent protection, improving the project’s economic feasibility and translational potential.

Refine

The fourth step, Refine, tightly connects the iHP cycle with our engineering cycle.

Stakeholder feedback is rapidly translated into experimental improvements and design optimization.

For example, during pathway design, we expanded our scope beyond naringin synthesis to include its precursor naringenin, enabling de novo synthesis.

These refinements not only advanced the scientific framework but also addressed stakeholder expectations for sustainable bioproduction.

(See Engineering Section for details.)

Recycle

After reflection and refinement, we reintroduced the improved designs to our original stakeholders, initiating a new round of the iHP cycle.

For example:

• When we presented our biosafety-enhanced probiotic design to early-onset CRC patients, they expressed greater willingness to try it.
• When we showcased our acid-sensitive expression system to clinicians, they affirmed its potential to better match the tumor microenvironment.

Through this repeated cycle, our project continuously strengthens its scientific validity, biosafety, and social value, evolving into a responsible, feasible, and forward-looking solution.

By coincidence, one of our team members accompanied his grandfather during colorectal cancer (CRC) treatment and noticed that his grandmother prepared Chenpi (dried tangerine peel) soup every day.

As a common traditional Chinese medicine (TCM) ingredient, Chenpi is widely used for regulating Qi, improving digestion, and strengthening the spleen.

This simple observation sparked a question that eventually shaped our project:

Does Chenpi have scientific evidence or potential therapeutic roles in CRC treatment and recovery?

Direction Confirmation

Traditional Chinese Medicine

Through extensive literature review, we found that Chenpi may exert anti-cancer effects by modulating gut microbiota, thus influencing CRC progression.

To validate this insight, we consulted TCM experts, which refreshed and deepened our understanding.

Both literature and interviews revealed that Chenpi exhibits anti-inflammatory, antioxidant, and digestive-promoting properties.

This evidence helped us confirm the potential of Chenpi as an adjuvant in CRC management.

By cross-comparing expert opinions and research data, we verified Chenpi’s role in regulating intestinal inflammation and improving gut homeostasis, which guided us in defining our research focus.

However, we also realized a practical limitation: traditional extraction methods result in low yield and poor stability of active compounds.

This insight led us to consider engineering microbial synthesis as a novel solution to overcome this bottleneck.

Natural Product Expertise

Our literature review revealed that Chenpi contains multiple classes of bioactive compounds, including flavonoids, volatile oils, and polysaccharides.

These constituents collectively contribute to Chenpi’s complex pharmacological activities, particularly in Qi regulation, anti-inflammation, and antioxidation.

In further investigations, we interviewed natural product researchers, who pointed out that among all these compounds, Neoeriocitrin stands out as one of the most potent and well-characterized active components, known for its anti-inflammatory and antioxidant properties.

We recognized that attempting to reconstruct all Chenpi compounds would make the project overly complex and unfocused, preventing us from achieving meaningful outcomes within the iGEM timeframe.

Therefore, we strategically chose to focus on Neoeriocitrin as our core molecular target.

By reconstructing the biosynthetic pathway of Neoeriocitrin in engineered E. coli, we aim to:

1. Verify its functional relevance in CRC prevention and therapy; and
2. Overcome the limitations of low yield and high extraction cost in traditional methods.

This approach provides both scientific validation and a scalable production route, paving the way for future clinical translation and product development.

Needs Identification

Questionnaire Survey

We first designed and distributed a questionnaire focusing on the incidence trend of colorectal cancer (CRC), lifestyle and cancer knowledge, public understanding of Chenpi, and the potential value of neoeriocitrin in treatment.

The results showed that most respondents were aware of the rising incidence of CRC but lacked understanding of early-onset CRC and its risk factors; they had some awareness of Chenpi, but mainly as a “traditional health food”; almost no one knew about neoeriocitrin.

[keywords: public awareness, early-onset CRC, Chenpi cognition, neoeriocitrin perception]

We systematically recorded and analyzed the results:

• Public level: limited knowledge of CRC prevention and treatment, especially regarding early-onset cases.
• Cognitive gap:Chenpi was commonly perceived as a dietary supplement, with few people aware of its pharmacological basis. These findings provided us with an initial social perspective, helping to clarify the key areas for future project implementation. However, we realized that the questionnaire alone did not yield sufficient insights to inform our technical design.

We reflected that a questionnaire could not capture patients’ real experiences, clinicians’ professional judgment, or potential application risks. Especially in terms of safety and feasibility, more in-depth primary data were needed.

This led us to the conclusion that one-on-one interviews would be essential for gathering more meaningful and actionable feedback.

CRC Patients

CRC patients are our primary stakeholders. Through direct communication, we deeply understood their suffering during treatment, particularly the severe side effects of radiotherapy and chemotherapy.

This experience made us realize that while traditional therapies can prolong life, they greatly reduce quality of life. We began to explore whether engineered probiotics could provide a gentler and safer form of adjuvant therapy. During interviews, patients expressed concern about the side effects of current treatments but also showed cautious optimism toward new therapies. They hoped for less toxic, more affordable, and easier-to-use alternatives. We documented their responses and identified these as core needs that our project must address. We also realized that discussing patients’ medical details too deeply could cause psychological stress. Therefore, our interviews focused more on daily experiences and unmet needs rather than clinical specifics. This reflection reminded us that our technology must also respect patients’ emotional comfort and acceptability. After gathering patient feedback, the idea of using probiotics as a therapeutic platform emerged. We then interviewed patients’ families, who provided additional insights from economic and emotional perspectives—such as treatment cost, caregiving burden, and future expectations. These conversations helped refine our project’s positioning: to consider not only efficacy, but also economic impact, usability, and family burden.

Family Members of Patients

In interviews with family members, we learned that besides treatment outcomes, they are more concerned with treatment costs and time spent caregiving. They expressed a strong demand for low-cost and time-saving therapies, and emphasized the importance of cancer prevention. This made us realize that the project should consider economic feasibility and convenience, in addition to therapeutic efficacy.

They hope for a low-cost, convenient therapy to reduce family burden. They also value preventive measures, believing that if probiotics can regulate the gut environment to reduce cancer risk from the source, it would be of great value.

We recognized that family feedback mostly focuses on economic and daily life aspects, whereas clinical and professional medical information is relatively limited. This suggests that subsequent research should focus on doctors, to obtain more professional opinions and supplement understanding of feasibility and safety of the therapy.

Doctors

During interviews with doctors, we further learned that CRC incidence continues to rise, especially early-onset cases. Doctors noted that CRC causes are complex and closely related to dietary habits, genetic factors, and gut environment. This highlighted the broad societal and medical demand for new therapies.

On one hand, existing treatments are effective but have high side effects and costs, affecting long-term quality of life. On the other hand, doctors suggested that if gut microbiota modulation could be used for prevention or adjunctive treatment, it would be highly valuable.

Based on doctors’ recommendations, we clarified the direction for subsequent design: combining Neoeriocitrin synthesis with a probiotic delivery platform, positioning it as a therapy that can provide both adjunctive treatment and preventive potential.

Feasibility

In discussions with gut microbiota experts, we learned that gut dysbiosis is one of the key factors inducing colorectal cancer (CRC). This insight made us realize that relying solely on traditional treatments cannot fundamentally improve the microecological environment, and that modulating the gut microbiota could be a novel breakthrough.

Experts pointed out that Neoeriocitrin from Chenpi has significant gut microbiota regulatory functions and can improve the inflammatory environment, thereby playing a role in cancer prevention and therapy. If Neoeriocitrin could act directly on the gut microbiota, then using engineered probiotics to produce and release Neoeriocitrin in situ would be not only scientifically feasible but also enhance targeting and application value.

We optimized our project design: establishing engineered E. coli synthesis of Neoeriocitrin as the core pathway, creating a platform capable of simultaneously modulating the gut microbiota and preventing CRC.

First-Generation Strain – Hesperetin to Neoeriocitrin

Based on extensive literature review and referencing previous iGEM teams’ designs, we confirmed that Neoeriocitrin synthesis relies on the precursor Hesperetin, and constructed a three-step glycosylation pathway:

• UDP-rhamnose synthesis: Introduce VvRHM-NRS from grape to convert UDP-glucose into UDP-rhamnose.
• 7-O-glucosylation: Use UGT73B2 from Arabidopsis to convert Hesperetin into Hesperetin-7-O-glucoside.
• 1,2-rhamnosyl transfer: Catalyzed by Cm1,2RhaT from grapefruit to generate Neoeriocitrin.

We recognize that while this design allows the first de novo synthesis of Neoeriocitrin in E. coli, breaking the limitations of natural extraction, it still depends on the Hesperetin precursor, which limits yield and stability. We plan to consult experts in metabolic engineering and experimental design to further optimize the research direction.

Second-Generation Strain – Naringenin to Neoeriocitrin

Through discussions with synthetic biology mentors, we realized that to truly develop the project as a probiotic therapy, the engineered strain must have the ability to synthesize Neoeriocitrin de novo. This led us to revisit the limitations of the first-generation design.

Based on this feedback, we decided to expand the synthetic pathway experimentally to break the dependency on Hesperetin. We began attempting the Naringenin-to-Neoeriocitrin pathway as a critical step toward full de novo synthesis. By introducing key enzymatic systems into E. coli, we aim to gradually construct a complete metabolic pathway, laying the foundation for future probiotic applications.

Acid-Sensitive Expression System

Discussions with metabolism research mentors revealed that acidic environments are a hallmark of the tumor microenvironment (TME). Due to abnormal tumor cell metabolism, lactic acid accumulation (the Warburg effect) lowers pH, promoting tumor invasion and metastasis while inhibiting immune cell function, reducing immunotherapy efficacy. This indicated that targeting acidic microenvironments is an important direction in anti-tumor research.

Mentors suggested using E. coli acid-inducible CadBA promoter, which is activated at pH < 5.8, allowing specific downstream gene expression in the tumor acidic environment. This provides a molecular switch to control Neoeriocitrin synthesis. Continuous expression could impose metabolic burden or cause off-target effects, while the acid-sensitive system improves targeting and minimizes effects on normal tissues, enhancing safety and controllability.

Based on this concept, we inserted rhamnosyl transferase (Cm1,2RhaT) under the CadBA promoter, so that Neoeriocitrin synthesis is specifically activated in the acidic environment of CRC, achieving efficient, targeted, and controllable therapeutic effects.

Project Safety

Safety is a core iGEM principle. During project design, constructing biological safety systems is paramount. Discussions with doctors highlighted that due to altered gut environments in some patients, probiotics may pose unexpected side effects, emphasizing the need for both efficacy and safety.

Based on this, we first designed an arabinose-inducible suicide system, allowing engineered strains to self-destruct in vivo upon exogenous arabinose addition, preventing potential harm.

However, we realized that in vivo safety alone is insufficient. If engineered strains leak into the external environment, they could pose ecological risks. Therefore, we added a cold-inducible suicide system: when strains leave the body and encounter low-temperature environments, the system triggers automatic lysis, achieving dual in vivo and ex vivo safety protection.

Patient Revisit

After the project reached its preliminary stage, we revisited colorectal cancer (CRC) patients and introduced them to the basic functions of the product as well as the dual safety system. After hearing the explanation, patients expressed willingness to try our engineered probiotic solution. They generally hoped that the product could be launched as soon as possible and significantly reduce treatment costs.

Patients’ expectations not only focus on scientific breakthroughs but also on the speed of clinical translation and economic affordability. This reminded us that the next step of the project must invest more in translation efficiency and cost control.

Family Revisit

We also revisited the family members of CRC patients, focusing on the cancer-preventive functions of the product. The families expressed a positive attitude and willingness to try it. They believed that if the product could prevent cancer onset, it would greatly reduce both the financial and emotional burden on families.

Adjunctive therapy and preventive value are key concerns for families. This indicates that the project could serve not only as an adjunct therapy for CRC patients but also has potential for cancer prevention in healthy populations.

To ensure that those in need can truly use our product, continuous design optimization and production cost reduction are necessary. At the same time, we need to explore the commercialization path, promoting the product from laboratory to market. Only by combining scientific validation with industrial translation, along with effective promotion and science communication, can our solution achieve broader real-world application, benefiting both patients and the public.

Product Formulation Determination

To enable the product to be applied in clinical and daily life, we must consider the formulation of the engineered probiotic. Discussions with probiotic powder manufacturers revealed that freeze-drying technology is currently the main method for probiotic preservation and application, significantly enhancing strain stability and convenience.

The manufacturers explained the production process and key steps, including fermentation culture, centrifugation, freeze-drying, and quality testing. They emphasized that freeze-dried formulations not only extend shelf life but also maintain bacterial viability, making transportation and use easier. If the product remained only in liquid or laboratory form, its promotion and application would be limited by storage conditions and transport costs. Freeze-dried formulations solve these issues, enhancing both practicality and industrial potential.

Based on this feedback, we decided that in subsequent experiments and product design, freeze-dried powder will be the preferred final delivery form, and we will explore adding protectants during freeze-drying to ensure that the engineered strain can efficiently synthesize Neoeriocitrin after reconstitution.

Intellectual Property & Regulatory Pathway Interviews

As our project gradually moves toward application and translation, we realized that intellectual property protection and regulatory compliance are two key pillars for the real-world implementation of research outcomes. To gain a comprehensive understanding of the potential path for engineered probiotic products—from technical protection to market access—we interviewed two experts: one in intellectual property (IP) and another with experience in the FDA approval process.

Intellectual Property Protection

The IP expert indicated that the core innovation of an engineered bacterial project usually lies in genetic circuit design and application scenarios, which can be fully protected through patents. They emphasized that different countries vary in their IP protection scope for synthetic biology and probiotic products: some focus more on functional innovation, while others emphasize safety and ethical review. This feedback made it clear that in future patent applications, we should focus on defining patentable boundaries and technical protection strategies.

Based on these suggestions, we decided to consider patent layout and result protection early during project planning and outcome presentation, ensuring that our research achievements can be legally and systematically protected, providing a foundation for productization and commercialization.

Regulatory & Approval Pathway

The FDA-related expert provided crucial guidance from a regulatory perspective. They explained that the FDA applies differentiated approval mechanisms for different types of biological products:

• New drug products must undergo full preclinical studies and clinical trials.
• Probiotic or engineered bacterial products may apply as dietary supplements or medical foods, which have simplified approval pathways, but still require rigorous safety and efficacy validation.

This highlighted that to truly bring a research outcome to market, laboratory validation alone is insufficient. Regulatory compliance, data support, and safety assessment are core elements for product translation.

Interview Insights

From these interviews, we formed a preliminary three-step plan for future productization:

• Safety verification – ensuring biosafety and environmental controllability of engineered strains.
• Efficacy evaluation – using data to prove expected effects and stability.
• Regulatory compliance – aligning with regulatory standards early in the development process.

These interviews clarified both patent and approval boundaries, making the team realize that moving from scientific discovery to social application requires integrating innovation, compliance, and safety for synthetic biology outcomes to truly reach the real world.

Commercialization Pathway

Investors

Commercialization is critical for translating the product into real-world use. We interviewed investors to obtain professional advice on business models, market entry points, and financing strategies. During interviews, we introduced the product design concept and business plan. Investors provided specific advice on market positioning, cost control, promotion channels, and potential competitors, and expressed willingness to support us at the appropriate stage.

Based on this feedback, we plan to further refine the business plan, define target user groups, optimize cost structure, and explore potential industry partnerships.

Insurance Companies

Economic accessibility is key to ensuring that patients can truly benefit from the product. Even if the technology is mature, high prices can make it unaffordable. We interviewed insurance companies to understand the current costs of cancer treatment and coverage under public and commercial insurance. They explained the average cost of cancer treatment, patient coverage rates, and conditions for drug inclusion in insurance systems. They also discussed the possibility and challenges of including engineered probiotic products in insurance reimbursement.

Based on this feedback, we incorporated insurance and reimbursement considerations into the business plan and plan to continue communication with relevant institutions to ensure the product is both safe, effective, and economically accessible.

Government

We have had preliminary exchanges with local government departments and regulators to understand their attitudes toward CRC prevention, probiotic regulation, and translational applications. These discussions provide important references for project compliance and application prospects.

However, we have not yet conducted in-depth discussions with core regulatory agencies such as the National Medical Products Administration (NMPA), nor do we have a systematic understanding of international differences in engineered probiotic and synthetic biology drug regulations. In the future, we will continue to expand dialogue with national and international regulators to ensure the project meets strict regulatory requirements.

Overall, our human practice journey demonstrates our continuous reflection and exploration on how synthetic biology can address major medical and social challenges. By actively engaging with a wide range of stakeholders—from patients and families to doctors, researchers, and policymakers—we continually refine our understanding of unmet needs and translate them into feasible designs.

Addressing shortcomings in current therapies—such as high costs, severe side effects, and limited accessibility—we aim to propose a solution that is safer, more economical, and sustainable. By adhering to the core values of synthetic biology—integrating natural product biosynthesis, engineered probiotics, and biosafety systems—we strive to bridge the gap between laboratory innovation and clinical application.

Our journey does not stop here. With the relentless passion of high school students, we will continue pursuing innovative paths, bringing hope to patients, reducing family burdens, and promoting synergy between technology and humanity for a better future.