“The true value of scientific innovation lies not only in how it is understood and accepted by society, but, more profoundly, in its ability to alleviate authentic human suffering.”

Postoperative residual disease in colorectal cancer (CRC) is the primary culprit behind a recurrence rate of up to 30% in stage II-III patients. Traditional systemic chemotherapy and radiotherapy carry significant side effects, forcing patients to compromise their quality of life (QoL) while fighting the disease. Our RectomeDy FotoZymogen (RDFZ) project stems from a deep insight into this clinical contradiction: the conflict between eradicating the lesion and preserving the patient’s QoL.

Our RDFZ-CHINA team adopts Human Practices (HP) as the first-principles guide for the entire project lifecycle. Starting with the experience of a former teacher who had to resign due to the debilitating side effects of chemoradiotherapy, we designed an innovative auxiliary treatment system based on engineered probiotics that achieves red-light-inducible, localized, and precise lesion clearance. To date, we have conducted over 40 interdisciplinary consultations spanning clinical practice, scientific research, industry, and regulation, ensuring our system is a responsible, trustworthy, and patient-centric solution.

The inspiration for our project comes from the personal experience of our teacher, Mr. X, who was well known for his engaging and passionate teaching style. After being diagnosed with colorectal cancer, which was too close to the anus for preservation, he underwent an abdominoperineal resection and a permanent colostomy, and resigned from his job. As we visited him, he shared the struggles he faced after the surgery. The colostomy introduced frequent complications like parastomal hernia, which reduced the sealing of the stoma bag, leading to leakage of feces. The psychological burden of managing the colostomy also made him avoid social interactions, as he feared judgment from others and worried about potential leaks or unpleasant odors associated with the stoma bag. Similar complications faced by colorectal cancer patients highlights the urgent need for non-invasive treatment systems for colorectal cancer.

Note：Beyond the initial inspiration provided by "Teacher X," whose experience is shared with their express consent, we will not disclose the specific names, portraits, or any identifiable personal information of subsequent cancer patients, family members, or other sensitive stakeholders involved in our communication processes, including questionnaire feedback, in-depth interviews, and clinical consultations.

As emphasized by the core spirit of iGEM, the essence of an excellent project is its ability to assimilate feedback from the external world, ensuring that innovation is responsible, effective, and safe. For RectomeDy FotoZymogen, a groundbreaking live biotherapeutic product (LBP), its application involves the complex dimensions of biosafety, clinical ethics, and public acceptance.

Therefore, we meticulously evaluated all potential stakeholders using the Mendelow’s Matrix (Power-Interest Matrix). Through this systematic framework, we categorized groups based on their Ability to provide important feedback (Power) and their level of concern for the project’s progress (Interest), enabling us to formulate targeted two-way communication and integrated strategies.

By assessing the power and interest of all stakeholders, we ensured that the project's design, safety, and future implementation possess social responsibility and high feasibility.

1、High Power - High Interest:

We categorized government regulators (e.g., NMPA officials), biosafety committee experts, and oncologic surgeons as "Key Players" (High Power - High Interest). Their feedback directly drove core decisions regarding the project's responsibility and clinical feasibility. For instance, it was the negative feedback from biosafety experts that forced us to abandon the uncontrollably toxic hemolysin and pivot to the safer PD-L1 nanobody (nb) immune strategy. Similarly, input from clinicians directly led to the inclusion of the HlpA targeting module to address the critical clinical challenge of intestinal peristalsis.

2、High Power - Low Interest:

For high-power, low-interest groups such as bioethicists, life science lawyers, and drug delivery specialists, we employed a "Keep Satisfied" strategy to ensure compliance and technical optimization. Consultations with lawyers prompted us to proactively draft management recommendations for the clinical application of engineered microbes, fulfilling our social responsibility as innovators. Recommendations from drug delivery experts solved the engineering bottleneck of low PD-L1 nb secretion efficiency, ultimately achieving effective payload release by adding the YopE signal peptide.

3、Low Power - High Interest:

Concurrently, we adopted an "Inform Fully" strategy for low-power, high-interest groups—CRC patients, their families, and frontline nurses. Although they formally lack the power to veto the project, their feedback is the core driver of user experience and safety trust. Patients' deep-seated concerns about residual live bacteria drove the design and construction of our multiple-redundancy suicide system. Feedback from nurses on ease of operation propelled the hardware's iteration from a two-step procedure to a one-stop (magnetically controlled capsule) delivery, maximizing patient adherence.

4、Low Power - Low Interest:

Finally, low-power, low-interest groups, such as the general media and K-12 students, were our "Minimum Effort" focus. For these groups, our primary action was to conduct Public Engagement (PE) activities to disseminate knowledge of synthetic biology and advocate for the concept of responsible innovation, achieving goals of popular science education and public awareness.

Throughout our Integrated Human Practices (IHP) journey, we engaged in robust two-way interaction with these four categories of stakeholders. We synthesized and analyzed their invaluable suggestions, distilling them into three core dimensions that profoundly impacted our RectomeDy FotoZymogen project.

Dimension One: Necessity & Clinical Significance

Through in-depth interviews with CRC patients and frontline oncologists, we concretized the brutal clinical contradiction of "high recurrence rate coupled with systemic toxicity" in auxiliary therapy. Mr. X’s suffering inspired our firm conviction that the goal of “low side effects” must supersede mere killing efficacy. These exchanges fully demonstrated the urgent need and significant value of our innovative red-light-inducible, localized, and precise clearance therapy, which forms the foundation of all our subsequent design.

Dimension Two: Science, Safety & Ethics Compliance

This is the area where HP feedback most intensely drove core project iteration. The negative feedback from the Biosafety Committee experts directly led to a fundamental shift in our therapeutic mechanism—abandoning the uncontrollable hemolysin in favor of the safer immune activation (PD-L1 nb) strategy. Furthermore, to address patients' profound concerns about residual live bacteria, we integrated a multiple-redundancy MazF/MazE suicide system. On the ethics and regulatory front, we proactively consulted legal experts and attempted to draft preliminary management recommendations for the clinical application of genetically engineered microbes, demonstrating our commitment to responsible innovation.

Dimension Three: Implementation & User Experience

Our considerations for the project never stopped at the laboratory bench. Addressing the challenge of intestinal peristalsis raised by clinicians, we incorporated the INP-HlpA targeting and anchoring module to ensure high therapeutic efficacy in the complex gut environment. More importantly, through repeated communication with patients, doctors, and nurses, we underwent three generations of hardware iteration: from the initially uncomfortable two-step procedure, to the second generation with stimulating edges, and finally designing a magnetically controlled, smooth, one-stop, spherical capsule for drug delivery. This iterative history fully illustrates our effort to integrate "user-friendliness" into the design, quantified and controlled precisely by the OptoDoseModel.

Our project progress was not linear but rather a constant spiral of iteration and ascent within a rigorous Reflective Cycle. Every interaction with a stakeholder served as a new "Impetus", driving deep reflection and optimization in every step of our Design-Build-Test-Learn (DBTL) process.

Our project's success is rooted in the continuous execution of the NICCI Reflective Cycle, which efficiently translates external feedback into internal design improvements.

Need: Defining the Real-World Problem

Dimension	Reflection Content
Problem Identification	What were the initial social, clinical, and environmental pain points we identified?
Existing Solutions	What are the current solutions? What are their limitations?
Users/Target Audience	Who are our ultimate users or beneficiaries? How did we determine their genuine needs for the solution?
Value Proposition	What unique, indispensable value does our project offer?

ur project was fundamentally driven by the imperative to alleviate authentic human suffering. The initial and most pressing need, validated by a large-scale questionnaire and the personal experience of a suffering teacher, was to develop a low-side-effect, more humane auxiliary treatment for colorectal cancer (CRC). Traditional systemic therapies and colostomy procedures resulted in high recurrence rates and severely compromised patients' quality of life. As the project progressed, the needs became technical and complex: we had to address the crucial biosafety challenge of the therapeutic regimen, which demanded replacing the toxic hemolysin; ensure therapeutic efficacy by solving the low secretion efficiency of the PD-L1 nanobody (nb); achieve precise control over drug release to avoid systemic toxicity; develop a method for targeted bacterial anchoring to counteract perpetual intestinal peristalsis; and, finally, meet our social responsibility by designing a multi-redundancy suicide system for safe clearance and addressing the existing regulatory gaps for live biotherapeutics. In the hardware phase, the core need shifted to user experience: designing an integrated, high-adherence drug delivery system that could safely and comfortably deliver both the engineered bacteria and the red light source in a one-stop, non-irritating procedure, solving issues like excessive device diameter, limited patient comfort, and complex two-step administration common in first- and second-generation prototypes.

Dimension	Reflection Content
Expert Interviews	Which experts in key fields did we consult? What substantial, project-changing advice did they provide?
Public Outreach/Education	What scientific knowledge did we disseminate to the public, students, or specific communities? How did we measure the effectiveness of these activities?
Cross-Team Collaboration	Did we collaborate with other teams? What were the specific contents and outcomes of the collaboration? How did this collaboration solve challenges specific to our project?
Feedback Loop	What mechanisms did we establish to gather and integrate community feedback?

Every major breakthrough in our project stemmed from a critical spark of inspiration often rooted in empathy and cross-disciplinary expertise. The foundational inspiration was the teacher's struggle, which instilled the non-negotiable mission of developing a low-side-effect, humane therapy. During the scientific deep dive, the inspiration for mechanism transition came when experts suggested the PD-L1 nanobody as a safer “immune activation” direction after the initial hemolysin idea failed. To fix the subsequent secretion problem, the concept of the YopE signal peptide as a "high-efficiency secretion leader" provided the precise engineering solution. When seeking drug control, the limitations of traditional systems (pH/temp) led to the crucial red light induction idea via the NETMAP system, valued for its penetration and biocompatibility. The challenge of intestinal motion was solved by drawing inspiration from HlpA targeting HSPG and the robust scaffold function of Ice Nucleation Protein (INP) to create the INP-HlpA anchoring system. Even safety design was inspired by the MazF/MazE toxin-antitoxin system for redundancy. Finally, in hardware, initial ideas for suppositories led to the practical inspiration of using magnetic switches for waterproof control, while feedback from a nurse manager provided the ultimate ergonomic inspiration to adopt the spherical light core and bullet-shaped geometry, replacing uncomfortable flat designs and prioritizing patient comfort.

InterCommunication: The Engine of Integrated Human Practices (IHP)

Dimension	Reflection Content
Expert Interviews	Which experts in key fields did we consult? What substantial, project-changing advice did they provide?
Public Outreach/Education	What scientific knowledge did we disseminate to the public, students, or specific communities? How did we measure the effectiveness of these activities?
Cross-Team Collaboration	Did we collaborate with other teams? What were the specific contents and outcomes of the collaboration? How did this collaboration solve challenges specific to our project?
Feedback Loop	What mechanisms did we establish to gather and integrate community feedback?

Our commitment to Integrated Human Practices (IHP) was demonstrated through continuous two-way communication that fundamentally shaped the project. Initial communication involved distributing over 200 valid questionnaires to validate the need and establish dialogue with CRC patients and their families. In the scientific phase, IHP was intensely integrated: the iGEM Safety Committee's negative feedback served as a critical design constraint, compelling the replacement of hemolysin. Consultations with a Peking University drug delivery specialist twice solved major engineering bottlenecks, first by adding the YopE signal peptide and later by suggesting the red light induction system. Multiple dialogues with Dr. Cheng Rui from the National Center for Digestive Diseases provided clinical validation and highlighted the intestinal peristalsis challenge, which directly led to the INP-HlpA module. Further patient engagement highlighted concerns about residual live bacteria, driving the multi-redundancy suicide system design. Recognizing regulatory gaps, we proactively consulted legal experts and drafted management recommendations, embodying social responsibility. Finally, in the implementation phase, consultation with an engineering expert addressed manufacturing, and crucially, an in-depth two-way interview with a nurse manager provided invaluable frontline user feedback on irritation risks and complexity, directly prompting the final ergonomic, one-stop, spherical hardware iteration to maximize patient adherence.

Critical Analysis: The Principle of Rigorous Self-Correction

Dimension	Reflection Content
Challenges and Obstacles	What were the main scientific or engineering challenges encountered during the project? How did we overcome them?
Lessons from Failure	Which experimental failures or design flaws ultimately prompted crucial design iterations? (i.e., What did we learn from failure?)
Biosafety and Ethics	What potential biosafety or ethical risks exist in our project? What specific measures did we take to mitigate or eliminate these risks?
Models and Data	Is our mathematical model or computational analysis rigorous? Do the experimental data support our core hypothesis?
Reproducibility	Are our experimental protocols detailed enough to ensure other laboratories can successfully reproduce our results?

Critical analysis served as our project's built-in mechanism for rigor, safety, and self-correction, ensuring the project constantly spiraled toward optimal design. The initial analysis established the safety-over-efficacy principle. This rigorous thinking led to the most fundamental mechanism shift in the scientific phase: immediately and critically recognizing the contradiction between hemolysin's uncontrollable toxicity and our mission, resulting in its swift replacement with the PD-L1 nb. We critically analyzed experimental failures (low PD-L1 nb secretion) and identified the fault not as gene expression but as a secretion pathway defect, guiding the implementation of YopE. Furthermore, we critically evaluated the limitations of traditional induction systems (pH/temp) in the challenging gut environment, leading to the selection of the red light system. We also critically ensured redundancy in biosafety, designing a multi-layered MazF/MazE system to mitigate the failure of a single mechanism. Finally, the critical analysis of the first- and second-generation hardware exposed clinical risks (mucosal irritation from the flat shape) and poor patient adherence (complex two-step process), prompting the final shift in design focus from mere functionality to uncompromised comfort and usability in the spherical, bullet-shaped capsule.

Implementation: Translating Design into Deliverables

Dimension	Reflection Content
Technology Maturity	What development stage is our technology currently in? What key technical challenges still need to be overcome before actual application or commercialization?
Scalability/Extensibility	Can our solution be applied or extended in different environments or to different targets? (e.g., expanding from colorectal cancer to other solid tumors)
Economic Feasibility	What are our production costs, delivery channels, and final pricing scheme? Is it economically competitive?
Future Planning	What are the defined goals for the next phase of our project? What roadmap have we established for the sustainable development of the project?

The implementation phase demonstrates our commitment to transforming conceptual ideas into tangible, tested deliverables. Early implementation involved the construction of the initial dual-payload system (Coa and Hemolysin plasmids) and the execution of CRC early screening education campaigns. Following critical analysis, we immediately implemented the fundamental mechanism switch by constructing the safer PD-L1 nanobody system and successfully engineered the fusion with the YopE1-15 signal peptide for enhanced secretion. We constructed the 660 nm red light-inducible NETMAP system and further implemented innovation by establishing a deep learning modeling and optimization framework for precise dose control. The challenge of intestinal motility was overcome by implementing the INP-HlpA surface display system. For biosafety, we implemented the multi-redundancy MazF/MazE suicide system and proactively drafted preliminary management recommendations for gene-engineered microbes. The culminating implementation was the three-generational hardware evolution: from the basic first-generation prototype to the structurally optimized second-generation suppository, culminating in the successful implementation of the final third-generation hardware model. This final model—a magnetic, spherical light core with a bullet-shaped body—achieved one-stop delivery, solving all previous user experience issues and validating the project as a comprehensive, safe, and patient-centric solution.

Need：

After visiting Mr. X, we deeply realized the physical and psychological harm caused by traditional colorectal cancer (CRC) therapies. The suffering endured by Mr. X during treatment and the profound impact on his quality of life convinced us that existing solutions sacrifice patients' humanized needs. This strong empathy for Mr. X drove our conviction to use synthetic biology to develop a more humane, low-side-effect treatment plan. We needed to determine if this case was an anomaly, whether this phenomenon was common, and what other needs CRC patients had beyond Mr. X's experience, requiring more samples, more data, more perspectives, and more feedback.

Inspiration：

With this conviction, we began our exploration. We decided to create a questionnaire to distribute to the public and consulted CRC patients we knew, inviting them to fill it out.

InterCommunication：

Out of the over 200 valid responses we received, approximately 45% of respondents came from individuals and families with potential treatment needs or high concern for CRC therapies. A significant portion of our subsequent communication with CRC patients or their families originated from this group of respondents. Among them, over 23% were troubled by personal experiences or the side effects and suffering witnessed in relatives. Concurrently, about 13% of participants had resolved the cancer threat itself through traditional methods like chemotherapy, radiotherapy, or surgery. Furthermore, 75% of respondents expressed deep concern about the side effects of chemotherapy and fear of the inconvenience caused by ostomy surgery, while 68% held both hope and suspicion regarding new technologies involving genetic engineering.

Critical Analysis：

A high proportion of the respondents who participated in our survey were troubled by traditional CRC therapies. We thus confirmed that Mr. X's needs and situation were not isolated cases, and the importance and universality of our project were validated. We held brainstorming sessions and collective learning discussions in our seminar, discovering that current CRC treatment primarily relies on surgical resection, supplemented by chemoradiotherapy to reduce recurrence and metastasis. It is the chemoradiotherapy and its subsequent side effects that cause Mr. X to continue suffering and distress after surgery, which is also the main concern of most questionnaire respondents. We began to read and reflect on literature related to cancer treatment.

Implementation：

By consulting existing literature on cancer therapies, we found that tumor growth relies on vigorous angiogenesis, and residual cancer cells possess strong environmental adaptability. Based on this, we decided to adopt a dual-payload therapeutic module collaborative strategy to achieve complementary mechanisms and more thorough clearance of cancer cells. Our initial design focused on the combination of "physical blockade" and "cell killing." To address the tumor's excessive demand for nutrients and oxygen, we chose prothrombinase (Coa) as the physical blockade module. Coa's principle is to catalyze the formation of a thrombus from fibrinogen, causing a physical occlusion of tumor blood vessels, thereby cutting off their supply of nutrients and oxygen. Experimental results confirmed the feasibility of this approach. Simultaneously, to specifically target and clear cancer cells, we paired Coa with a potent hemolysin, aiming to enhance the killing effect through its strong cell membrane lysis activity. In parallel, the questionnaire survey revealed a lack of public attention to CRC early screening. Therefore, we published a popular science article on CRC early screening on our official account and produced promotional tri-folds for public distribution, hoping to raise public awareness of CRC.

Communication with the iGEM Safety Committee

Need：

After outlining our preliminary solution ideas, we needed professional guidance to determine the feasibility of our method.

Inspiration：

We planned to send an email to the iGEM Safety Committee for inquiry and seek consultation with gastroenterology experts.

InterCommunication：

The Safety Committee sent an email, where the expert advised that hemolysin is not on the white list. It is a pore-forming toxin that can lyse red blood cells and other cell types. Direct exposure through skin contact, inhalation of aerosols, or accidental ingestion could lead to local tissue damage or systemic hemolysis, depending on concentration and route of exposure. Thus, we had to re-conceptualize our therapeutic module. We consulted biology experts to exchange ideas and seek advice. A biology expert suggested that the PD-L1 nanobody (nb) is an excellent means for cancer treatment. PD-L1 nb is a novel immune checkpoint inhibitor, extremely small in size, only one-tenth the size of a traditional antibody. Its primary function is to release the "brakes" of the immune system on the tumor. PD-L1 nb can block the binding of PD-L1 to PD-1, thereby re-activating T cells and NK cells to restore their ability to kill tumor cells. Due to its small size and high stability, PD-L1 nb is particularly suitable for efficient expression and release locally in the tumor microenvironment via engineered bacteria, achieving precise, low-toxicity tumor immunotherapy.

Critical Analysis：

Upon receiving the email from the Safety Committee, we realized that the goal of “low side effects”, inspired by Mr. X's experience, must supersede mere killing efficacy. We must not excessively pursue therapeutic effect while neglecting the inherent toxicity; the commitment to safety cannot be compromised. Consequently, we decided to replace hemolysin with PD-L1 nanobody, shifting our therapeutic module strategy to "physical blockade" combined with "immune activation."

Implementation：

We constructed plasmids for thrombin and PD-L1 nanobody and attempted to verify if they could function as anticipated.

Visit to Peking University Health Science Center

Need：

Western Blot experiments showed extremely low PD-L1 nb yield; we needed to solve this problem.

Inspiration：

We hypothesized that the issue might be a problem with the secretion step of PD-L1 nb, leading to a large amount of retention inside the cell, preventing effective secretion into the extracellular space to bind to tumor targets. We considered whether adding auxiliary factors could correct this.

InterCommunication：

This bottleneck led us to seek help from a drug delivery specialist at Peking University Health Science Center. The expert suggested adding the YopE1-15 signal peptide to PD-L1 nb. The YopE1-15 signal peptide, derived from the N-terminal 1 to 15 amino acid fragment of the Yersinia pestis YopE protein, can be used as a "high-efficiency secretion leader": when this very short peptide is fused to the N-terminus of the target therapeutic protein (such as the nanobody PD-L1 nb or other oncolytic drugs), it directs the fusion protein to the engineered bacterium's extracellular secretion pathway. This enables the bacteria to highly efficiently transport and release the therapeutic proteins they produce to the outside of the cell, entering the tumor microenvironment, thereby achieving localized and targeted drug delivery.

Critical Analysis：

We critically accepted the expert's advice and analyzed its scientific merit: given the time and risk of independently searching for a secretion leader, and that YopE1-15 is a well-validated, highly efficient secretion element, we determined this was a fast, accurate, and low-risk solution that must be immediately integrated into our design.

Implementation：

We immediately adopted the suggestion and constructed a PD-L1 nb fused with YopE1-15. Through Western Blot verification of the engineered bacterial supernatant, we confirmed that the signal peptide fusion design effectively resolved the secretion problem, thus locking in our final dual therapeutic payload. Simultaneously, we conducted the first round of communication with CRC patients after the survey. After introducing our therapeutic module idea, they raised a crucial question: How can we ensure these drugs are only released at the tumor site?

Visit to the Chinese Academy of Medical Sciences

Need：

After communication with cancer patients, we needed to address the controllability of the therapeutic module.

Inspiration：

We believed we could introduce an inducible system to control the release of the drug module. We brainstormed and discussed, concluding that traditional temperature, pH, or blue light systems all had limitations in the gut, such as low precision and insufficient penetration.

InterCommunication：

We communicated with the drug delivery specialist again and received a key inspiration: red light. She mentioned that the current NETMAP (Near-Infrared Light-Mediated PadC-based Photoswitch) system has a "light-chemistry-transcription" signal conversion chain. The photosensitive element PadC converts the light signal into a biochemical signal. c-di-GMP, a second messenger unique to bacteria, acts as an intermediate, ensuring the high efficiency and specificity of signal transduction and reducing interference with host cells. The transcriptional activator MrkH ensures that the light signal can high-strongly initiate the transcription of downstream therapeutic genes. This system can utilize Near-Infrared (NIR) light, an external signal harmless to the human body and with strong penetration, to trigger gene expression.

Critical Analysis：

In live therapy, the ideal external signal must possess good tissue penetration and be non-toxic to the organism. We believed the red light induction system was more suitable for our project design. Furthermore, we realized that the NETMAP system allows us to quantitatively regulate the expression level of the therapeutic protein by simply adjusting the intensity or duration of red light exposure. We could use deep learning to build a forward prediction model, simulating the effect of different conditions on gene expression in the intestinal environment, and automatically inverse-derive the required light intensity to meet the target fluorescence level through numerical optimization methods, thereby providing a quantitative basis for in vivo experiments and light control hardware design.

Implementation：

We constructed the NETMAP system based on 660 nm NIR red light induction. Results proved that red light could successfully induce therapeutic protein expression. We also established a deep learning modeling and optimization framework for the intestinal light-controlled environment, proposing a light intensity inverse-derivation method based on numerical optimization, providing a feasible automated idea for optimizing experimental conditions.

Discussion with Dr. Cheng Rui from the National Center for Digestive Diseases

Need：

After successfully constructing the NETMAP system, we communicated with Dr. Cheng Rui from the National Center for Digestive Diseases. While affirming parts of our concept, she suggested that relying solely on the natural homing of engineered bacteria and red light activation might be insufficient to counter intestinal peristalsis. We needed to solve a new problem: How to achieve bacterial anchoring on the tumor surface?

Inspiration：

We prepared to introduce a targeting module. In a new round of literature review and collective brainstorming, we chose HlpA as the targeting domain. HlpA can specifically recognize and bind to Heparan Sulfate Proteoglycans (HSPG). HSPG is a key component of the extracellular matrix and is often overexpressed on the surface of CRC cells. HSPG overexpression is closely related to tumor proliferation, invasion, and metastasis.

InterCommunication：

We communicated with Dr. Cheng Rui again, and she affirmed our idea of HlpA. She advised us to consider how to counter the continuous peristalsis and flow rate of contents in the colorectum, as secreted HlpA might be diluted due to low local concentration, reducing therapeutic effect.

Critical Analysis：

After another collective discussion in the seminar, we chose to use the INP-HlpA surface display system. INP (Ice Nucleation Protein) acts as a natural outer membrane scaffold, achieving multivalent, high-density display of HlpA, providing high-strength physical anchoring.

Implementation：

The results showed that the addition of the adhesion system strongly ensured the basis for high therapeutic efficacy. This targeting module works synergistically with the red light induction module, jointly resolving the risk of drug leakage.

Participation in the National Synthetic Biology Innovation Competition as the Only High School Team in China

Need：

We conducted a second round of communication with CRC patients and their families, synchronizing our project's new progress and continuing to collect new suggestions and feedback. During the discussion, the topics of live bacteria residue and biosafety were particularly heated, forcing us to focus on the biosafety module. We needed a suicide system to ensure safety after the treatment is concluded.

Inspiration：

To achieve strict biocontainment and prevent the spread of engineered bacteria in a non-therapeutic environment, we designed a suicide system based on modular synthetic biology strategy, centered around the MazF/MazE toxin-antitoxin system.

InterCommunication：

We were invited to participate in Synbio Challenges competition held in Shenzhen, China, where we actively exchanged ideas and discussed with various experts and teachers. A comment from an audience member drew our attention: Beyond technical-level control, the existing regulatory system for the clinical application of engineered bacteria is lacking. Therefore, we proactively engaged in interdisciplinary learning, consulting legal experts to understand the current regulatory norms for live biotherapeutic products.

Critical Analysis：

Under the guidance of legal experts, we decided to attempt to draft preliminary management recommendations for the clinical application of genetically engineered microbes. We hoped to promote the improvement of relevant regulations and fulfill the social responsibility of innovators.

Implementation：

On the technical level, we designed a condition-responsive suicide system with multiple control redundancies, forming an "OR gate" logic: as long as either the red light or arabinose signal is present, MazE is expressed, neutralizing the MazF toxicity and ensuring the survival of the engineered bacteria. On the legal exploration front, we drafted a management recommendation. This action was approved by CRC patients.

While exploring our project methodology, we were also exploring hardware facilities.

Company Visit and Expert Consultation

Need：

We needed to explore a treatment method to deliver engineered bacteria to the colorectum and simultaneously deliver the red light source.

Inspiration：

After collective discussion, we rejected the traditional oral drug form and proposed using a suppository for drug delivery.

InterCommunication：

During communication with experts, we received an excellent suggestion: to use a magnetic switch to control the red light source, as manual mechanical switches were not suitable for our needs regarding water resistance and operability. Additionally, an expert suggested adopting a modular architecture, delivering the engineered bacteria and red light source separately. For engineered bacteria delivery, we could use a disposable syringe for pushing.

Critical Analysis：

We discussed the form of pushing the engineered bacteria with a disposable syringe, deciding that the gel-like engineered bacteria could be pushed. The external magnetic force for pre-activation could be achieved ex vivo using a Hall sensor. However, the electronic components of the red light source needed to be enclosed for safety. We discussed using epoxy resin to encapsulate the electronic components, adopting a cylindrical suppository shape.

Implementation：

We successfully produced the first generation of hardware device and presented our first-generation model at the Synbio Challenges competition in Shenzhen, China.

Hardware Field Research in Shenzhen

Need：

At the Synbio Challenges competition and during communication with patients and doctors, we received a great deal of feedback: the diameter exceeded 1 cm, limiting patient comfort and impacting clinical feasibility. The two-step delivery process of drug and hardware was overly complex, and the external form lacked ergonomic optimization.

Inspiration：

We decided to modify and upgrade the hardware, optimizing the three areas of concern raised by everyone.

InterCommunication：

We discussed and researched with an engineering expert, who explained the integrated molding process: production can be achieved by stepwise pouring and curing in a single mold for multiple materials. After the two materials cure, they are demolded together to obtain the final integrated product. This process avoids subsequent assembly gaps, forming a compact, structurally unified suppository.

Critical Analysis：

This exchange inspired our new idea: to integrate the design of our suppository. We would inject epoxy resin into the head region, integrating the red light source within the head. The gel-like engineered bacteria were not conducive to shaping, so we processed the engineered bacteria into lyophilized bacteria and used stearic acid as the shaping agent. Stearic acid containing the lyophilized bacteria would be injected into the tail region of the same mold. Concurrently, we changed the head-to-tail ratio of the suppository, reducing the head volume and enlarging the drug-loaded tail, focusing on ergonomics and usability.

Implementation：

We successfully iterated to the second generation of hardware device and engaged in extensive discussions with patients and doctors to better understand its practical clinical utility.

Communication with Medical Staff and Physicians

Need：

The second-generation suppository significantly improved ease of operation and usability. Patients were very interested, but when discussing the specific usage methods, we realized we needed to communicate with doctors and nurses with nursing experience.

Inspiration：

We invited Dashu Elderly Care Co., Ltd., one of Beijing's leading providers of integrated home elderly care services.

InterCommunication：

We invited a nurse manager with over a decade of practical experience in CRC patient care to our school for in-depth discussion. She first shared her experience in CRC patient care. We then shared our scientific concepts and showcased the second-generation suppository model, inquiring about its actual clinical utility. She offered very valuable suggestions and perspectives. Firstly, she pointed out that the flat resin surface exposed after stearic acid melts might irritate the intestinal mucosa, potentially causing scratches in the intestines or anus. Secondly, the external shape of the second generation still needed further evolution. Finally, the light source needed to be considered to adapt to the needs of CRC patients at different stages and severity levels.

Critical Analysis：

This collaboration provided us with an indirect but valuable clinical perspective, guiding us to be more meticulous in our hardware considerations, maintaining a patient-centric approach. We conducted the third generation of suppository iteration. We replaced the flat cylindrical head with a spherical epoxy resin light core and adopted a bullet-shaped geometric structure (head/tail made of stearic acid, spherical hardware in the center) to reduce insertion resistance and avoid sharp edges. We decided to offer versions with 2 / 4 / 6 / 8 LEDs without changing the overall structure, to meet different lighting intensity needs. We achieved dual-material stepwise pouring within a single mold, ensuring central alignment, consistent dimensions, and reduced failure rate. We also realized we should write an operation manual to guide patients and medical staff in its use.

Implementation：

We successfully iterated to the final version of the third-generation hardware model. Specific hardware information can be viewed on our hardware webpage.

The RectomeDy FotoZymogen (RDFZ) project illustrates how responsible innovation is born from fundamental empathy. Guided throughout by the NICCI Reflective Cycle, we successfully translated the suffering experienced by "Teacher X" into the final integrated suppository. The insistence on biosafety was our core driver: Critical Analysis led us to abandon uncontrollable toxins for the safer PD-L1 nb immune strategy. Two-way Communication directly integrated feedback from experts, patients, and nurses to solve the three major clinical challenges of secretion, anchoring, and adherence. The ultimate triumph of our project is the complete internalization of high ethical standards within the scientific design.

The core value of RectomeDy FotoZymogen lies in its platform potential and responsible innovation philosophy. Scientifically, we will focus on optimizing the OptoDose prediction model to ensure precise dosage control through computational modeling, and explore its application in other in vitro solid tumor models. Our commitment is to evolve the project into a scalable, high-safety technology verification platform. Simultaneously, we will continue engagement with academic and regulatory discussion forums, advocating for a new precision medicine standard centered on enhancing the patient’s quality of life, and encouraging deeper societal discussion on the ethics and regulation of engineered microbial therapies.