February 28, 2025-Prof. Baojun Wang

Genetic circuits are one of the core modules of our project, and the amplification & feedback elements in our initial design were developed by the research group of Professor Baojun Wang, director of the Institute of Synthetic Biology, ZJU-Hangzhou Global Scientific and Technological Innovation Center (HIC-ZJU). To this end, we visited the HIC-ZJU to consult with him on the design and optimization of the sensing module.

Prof. Baojun Wang also holds the position of Qiushi Chair Professor and Head of the Synthetic BioSystems Design Laboratory in the College of Chemical and Biological Engineering, Zhejiang University. With long-standing dedication to developing novel enabling technologies in synthetic biology, Professor Wang focuses on engineering orthogonal genetic circuits for the sensing and computation of multiple cellular and environmental signals. His research findings have been widely applied in fields including biosensors, biomanufacturing, and biotherapeutics.

After reviewing the project introduction, Prof. Wang emphasized the need to highlight the advantages of oral probiotics over semaglutide, particularly for oral tablet formulations. To enhance the genetic circuit's logic, he suggested replacing the original AND-gate with an orthogonal split intein developed by his research group[10]. Regarding hypoxic promoters, he noted that their characterization demands high technical expertise and substantial time. Thus, he recommended switching to an alternative inducible promoter for the localization system. Additionally, he pointed out that simultaneously implementing both gene circuit overexpression and protein modification could lead to potential side effects, such as excessive GLP-1 accumulation in vivo. Given the limited experimental timeframe, he advised prioritizing either the gene circuit design or the protein modification module to ensure timely and meaningful results.

Following Prof. Wang's recommendations, we replaced the original AND-gate elements with the coupled orthogonal split intein SspGyrB and split extracytoplasmic function (ECF) sigma factor σ16. We also obtained the plasmid E222, amplification element Amp30^E, and feedback gene hrpV, all constructed by his group. Furthermore, we conducted a literature review to screen a suitable replacement for the original hypoxic promoter. Prof. Wang's meticulous guidance on the gene circuit design module and objective assessments of each module have significantly strengthened our confidence and resolve to refine the circuit design.

Figure 7. Members of HiZJU-China consulting Prof. Baojun Wang

March 11, 2025-Assistant Prof. Nathan Crook

The intestinal microbiome is highly complex, and the impact of introducing exogenous engineered bacteria on system homeostasis must be carefully considered. To address this, we reached out to Assistant Professor Nathan Crook from the Department of Chemical and Biomolecular Engineering at North Carolina State University (NCSU) to discuss the optimization of our project. Prof. Crook's research group primarily focuses on microbial community engineering, utilizing high-throughput experiments and computational gene engineering techniques, with an emphasis on human gut microbiota.

During the discussion, Dr. Crook first pointed out that GLP-1 receptors are primarily located in the pancreas, small intestine, and other areas, whereas the intestinal regions naturally colonized by Escherichia coli do not have these receptors. Therefore, the main challenge lies in identifying how GLP-1 can effectively enter the bloodstream. He further added, based on his own experimental experience, that EcN can only persist in mice for a maximum of two days. In the absence of animal experiments, it is crucial to determine how to validate its colonization ability to ensure sustained GLP-1 production.

Regarding aspects related to expression levels in the project, Dr. Crook emphasized prioritizing and validating three key points: the effective secretion of GLP-1, the characterization of the PCA-responsive sensor efficiency, and whether normal green tea metabolites can induce sufficient GLP-1 production to meet therapeutic demands. Furthermore, he highlighted the importance of preventing excessive growth-related stress in E. coli following the introduction of the exogenous module, as this could compromise the strain's viability and functional stability.

Based on Assistant Prof. Crook's suggestions, we planned to optimize the design of the cell-penetrating peptide (CPP) to enhance the intestinal absorption efficiency of GLP-1, gradually test the optimal induction concentration and duration of the PCA-responsive sensor, and conduct experimental validation systematically by module. Afterwards, we will optimize the gene and plasmid design configuration, integrating data of all modules to provide robust data support for the project.

Figure 8. Online communication between HiZJU-China and Assistant Professor Nathan Crook

March 21, 2025-Dr. Nan Zhou

On March 21, recommended by a senior team member, we visited the HIC-ZJU to consult with Dr. Nan Zhou, a researcher at the Institute for Intelligent Bio/Chem Manufacturing. We discussed the theoretical research on gene circuit design for gut microbiota and its application in biopharmaceuticals. Dr. Zhou's research focuses on the fundamental studies and innovative applications of synthetic biology in biomedicine, using synthetic gene circuits to explore basic principles of life sciences and develop novel biopharmaceuticals.

After listening to our project presentation, Dr. Zhou highly praised the integrity of our existing modules but emphasized the need to focus on the core design. He noted that modifying GLP-1's sequence by incorporating non-canonical amino acids (ncAAs) poses greater challenges than designing the corresponding gene circuit, as it requires balancing enhanced activity and stability, and that improving quality and yield are not mutually exclusive. Regarding the secretion of GLP-1, he mentioned that due to its small molecular weight, it is difficult for E. coli to secrete effectively. He suggested either adopting tandem expression of multiple GLP-1 copies linked via enzymatic cleavage sites or employing GLP-1 fusion with other high-molecular-weight proteins for enhanced expression. In response to our idea of "using cell-penetrating peptides to promote GLP-1 transport across the intestinal mucosa," Dr. Zhou advised us to prioritize conceptual validation through literature reviews to compensate for our team's limited experience with animal experiments.

Furthermore, concerning the hypoxia-inducible promoter used in our original gene circuit, Dr. Zhou pointed out that conventional labs lack the equipment to create a hypoxic environment. He suggested replacing it with a bile salt biosensor as an intestinal localization system. Regarding the suitability of EcN, he shared his experimental experience that EcN was not suitable for in vivo colonization, even with gavage administration, and only remained in the mouse for 2-3 days. Moreover, it is difficult to engineer naturally colonizing Bacteroids in the short term. Therefore, he suggested that we need to further explore alternative bacterial strains. Finally, he mentioned that introducing a large number of exogenous plasmids into the chassis strain could lead to two problems: plasmid loss under low selective pressure, and an excessive metabolic burden that makes it difficult for the engineered bacteria to compete with the native flora in the gut.

Dr. Zhou's suggestions enabled us to recognize that for such a complex project, we must define the prioritization of our modules to highlight core strengths. Therefore, we decided to invest more effort into the GLP-1 sequence modification. At the same time, we adopted his suggestion to replace the hypoxia-inducible promoter with the bile salt sensing device (BBa_K1962010). While there is extensive literature supporting the use of EcN in treating intestinal diseases, and we are limited by our current laboratory conditions from switching to a better chassis strain, Dr. Zhou's feedback made us more aware of the insufficient colonization time. To address this, we plan to explore modified hydrogel carriers to enhance the adhesion and colonization capabilities of the engineered bacteria.

Figure 9. Communication between HiZJU-China and Dr. Nan Zhou

July 21, 2025-iGEM WLSA Shanghai Academy 2025

On July 14, HiZJU-China and WLSA Shanghai Academy iGEM team had a discussion on various aspects including project design, experiments, and progress. Both teams' projects focus on the same themes of weight loss and blood glucose reduction, and also shared a design centered on GLP-1. This created significant potential for mutual learning in aspects of experimental design and Human Practices.

During the specific exchange on experimental design, WLSA Shanghai Academy raised several core issues that might have been overlooked: how to quantitatively establish the relationship between protocatechuic acid (PCA) levels which correlate with the amount of green tea intake and GLP-1 expression; the regulatory effect of glucose intake on GLP-1 expression, with a focus on how glucose administration following hypoglycemia triggers the restoration of GLP-1 expression; the final effect of GLP-1 cell-penetrating peptides and the colonization efficiency of EcN; biosafety concerns related to dual-input suicide mechanisms, non-canonical amino acids, competition within gut microbiota and storage methods under temperature-sensitive kill-switch, etc. Additionally, they shared their HP activities with us, which included visiting hospitals for interviews with doctors and patients, creating team uniforms and souvenirs, designing social surveys to collect actual needs, producing animated science popularization videos, and establishing an official public account for diabetes education.

In response to suggestions from WLSA Shanghai Academy, we will refine the design of the gene circuit and characterize the induction threshold of the PCA-responsive sensor within this circuit. For the "HP" part, we will incorporate social surveys and physician interviews to collect real-world patients' needs and public acceptance of the project, ensuring the project is more aligned with reality.

Figure 10. Bridging and exchange between HiZJU-China and WLSA Shanghai Academy

February 25, 2025-Prof. Jianping Wu

For the protein engineering module, we initially consulted Professor Jianping Wu, the lecturer for our Protein Engineering course. Professor Wu is currently the Director of the Institute of Biological Engineering at Zhejiang University and the Deputy Director of the National-Local Joint Engineering Laboratory of Industrial Biocatalysis. He has long been engaged in research on biocatalysis, biotransformation, enzyme engineering, and biochemical reaction engineering.

After presenting the overall project design to Prof. Wu, he first recognized the maturity and feasibility of the project, but also highlighted that the project scope is overly broad, and we may encounter an excessive experimental workload within the limited timeframe. He recommended that we clearly define team roles and prioritize modules to push the project forward.

Regarding protein engineering, Prof. Wu suggested that the currently available biosynthetic pathways for non-canonical amino acids (ncAAs) are relatively limited, and since GLP-1 has a small number of amino acids, it was advisable to first conduct dry experiments to perform saturation mutagenesis and molecular docking simulations. Then, we should sequentially replace amino acids with ncAAs to assess the stability and activity of the mutants, prioritizing the maintenance of bioactivity while enhancing stability accordingly.

Additionally, he noted that if we introduce a substantial number of project-related heterologous genes and metabolic pathways into the E. coli strain, this could impose a significant metabolic burden on the bacterium. He advised validating each module separately first, then gradually optimizing the pathway expression and metabolic design.

Prof. Wu analyzed the potential issues that might arise during the experimental process from a practical perspective, and recommended treating protein engineering as the core module of the project, with specific division of labor. Considering the difficulty of conducting wet-lab animal experiments, he suggested that we begin with dry experiments such as molecular docking to perform preliminary exploration and help establish conditions for subsequent experiments. Based on this, we clarified the core direction for GLP-1 protein engineering and the entry point for the project, further refining the team's focus and division of tasks.

Figure 11. Communication between HiZJU-China and Prof. Jianping Wu

July 9, 2025-Mr. Ran Chao

As an alumna of Zhejiang University, co-founder and CEO of Hangzhou Yanjin Technology, Mr. Ran Chao has successfully developed original gene sequences for agricultural breeding by leveraging the independently built high-throughput protein design and testing platform. We were honored to have presented our project to Senior Ran Chao and received his suggestions.

After learning about our project, Mr. Chao commented that the project currently involves extensive experiments and literature research, and remains mostly in the stage of theoretical and technical verification, with significant challenges in in vivo experiments and high-throughput screening. He noted that the project had a complete plan and high innovation but was too complex, suggesting that we could prioritize verifying several core innovation points. Also, the practical regulatory response curve following oral drug administration will be a relatively striking point. Additionally, he recommended using high-throughput methods to accelerate module construction and optimization, and designing quantitative verification experiments to ensure module functions.

In light of Mr. Chao's suggestions, we have decided to refine our experimental plans and validate relevant designs: regarding the response of oral drugs, we will obtain more intuitive data support through experiments. Meanwhile, we will enhance our learning of high-throughput testing methods to apply dry lab experiments to the screening of protein modification sites.

Figure 12. Communication between HiZJU-China and Mr. Ran Chao

August 18, 2025-Dr. Zhikan Yao

GLP-1 is a small-molecule short peptide. Previously, following the suggestions of our senior team member, we used His-tag for adsorption via a nickel column, then enriched the protein through ultrafiltration tube centrifugation. However, no target band was detected by SDS-PAGE. We speculated that GLP-1 might have been eluted during ultrafiltration, resulting in an extremely low content of the target protein in the sample. Therefore, we consulted Dr. Yao Zhikan, whose research focuses on membrane science and technology, regarding issues related to protein enrichment.

Dr. Yao Zhikan is our lecturer of courses in Biological Separation Engineering and Environmental Biotechnology at our college, and currently serves as a distinguished researcher at the College of Chemical and Biological Engineering, Zhejiang University. His main research directions include the design and preparation of high-performance reverse osmosis membranes and nanofiltration membranes, as well as the application of novel membrane separation technologies in water treatment and resource utilization fields such as wastewater treatment, drinking water purification, and lithium extraction from salt lakes.

In response to our questions, Dr. Yao pointed out that given GLP-1 has a molecular weight of less than 10kD, we could try using nanofiltration membrane centrifugation. Meanwhile, during nickel column processing, increasing the dosage of raw material samples and imidazole eluent buffer to expand the solution volume, and performing high-pressure nanofiltration through repeated operations with the same membrane, could improve the enrichment efficiency of GLP-1. Additionally, Dr. Yao generously provided nanofiltration-related experimental equipment, along with technical guidance and protocols, offering significant support for the advancement of our project.

Figure 13. Group photo of HiZJU-China members with Dr. Zhikan Yao

August 18, 2025-Prof. Jianping Wu

The protein module encountered several technical challenges during the concentration and purification of GLP-1: due to the low molecular weight of GLP-1, it was hard to concentrate by ultrafiltration and prone to dispersion during protein electrophoresis, making it difficult to observe clear bands in in SDS-PAGE, which hindered subsequent purification and activity characterization. To address this, we once again consulted Professor Jianping Wu regarding experimental procedures.

To address the issues posed by GLP-1's low molecular weight, Prof. Wu proposed targeted improvement strategies. For protein concentration, he recommended enhancing post-purification GLP-1 concentration via freeze-drying, nanofiltration, and optimizing the imidazole concentration and flow rate of the elution buffer. For protein electrophoresis, he suggested using higher-concentration gels and molecular weight markers with narrower ranges to enhance the visibility of low-molecular-weight GLP-1 bands. These suggestions have provided valuable guidance for advancing wet lab experiments in the protein engineering module.

August 19, 2025-Dr. Hao Pan

On August 19, 2025, we held an online meeting with Dr. Hao Pan, Director of the Biological Analysis and Drug Metabolism Department at the Global Innovative Drug R&D Center, Huadong Medicine, to discuss technical details of the protein engineering module.

Dr. Pan is the project leader of HDM1002, a first-class innovative oral small-molecule GLP-1 receptor agonist independently developed by Huadong Medicine. This project has entered clinical Phase II trials, with promising prospects. We consulted Dr. Pan regarding the future development, market outlook, and core issues of preclinical research on drugs related to GLP-1 synthesis by E. coli. After developing a general understanding of the project, Dr. Pan opined that this project focuses on a hot topic in medicine, particularly in addressing the delivery challenges of injectable peptide drugs, which has long-term clinical and market value.

Dr. Pan pointed out that the core challenge of the project lies in verifying the pharmaceutical properties of modified GLP-1, specifically including their in vitro activity, membrane permeability, serum protein binding rate, and half-life extension efficacy. He recommended prioritizing experiments on in vitro binding activity and serum half-life. Moreover, Dr. Pan noted that clinically applied GLP-1-based drugs often exhibit significant gastrointestinal side effects, requiring gradual dosage escalation. Accordingly, the engineered bacterial expression system for GLP-1 must particularly prioritize dose controllability and the accommodation of individual differences.

In light of Dr. Pan's suggestions, we revised our strategy for testing the half-life of GLP-1. Initially, we planned to purchase NEP-24.11 enzyme to conduct in vitro enzymatic digestion experiments, with timed sampling to assess the degradation of GLP-1. However, relying solely on NEP-24.11 is insufficient to simulate the complex physiological environment in humans. We have therefore adjusted our approach to instead determine the plasma half-life of our GLP-1 using rat plasma, providing a more comprehensive evaluation of the modification effects. Beyond this, Dr. Pan's insightful guidance on protein modification and his reminder regarding drug side effects have significantly enhanced the overall development of our project.

Figure 14. Communication between HiZJU-China and Dr. Hao Pan

July 15, 2025-Dr. Yingfei Ma

During our summer camp at the Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, team members consulted Dr. Yingfei Ma, Deputy Director of the Synthetic Microbiome Research Center, regarding our project design. Dr. Ma's research group focuses on microbiomes, phageomes, phage synthetic biology, and the application of phages in combating drug-resistant bacteria. Given our plan to apply for awards in Safety and Security, we sought his advice on how to integrate specific targeting phages into the regulatory systems of this section.

Dr. Ma first noted that natural E. coli accounts for a low proportion in the human gut, making it unsuitable for intestinal colonization and large-scale drug production. Thus, he also recommended using Bacteroides as the chassis, though this would involve a relatively longer experimental cycle. Regarding phage applications, he emphasized that phages exhibit strain-specific targeting. The GLP-1 release system designed based on phage lysogenic conversion and bacterial lysis is more efficient than secretory expression systems via signal transduction of lysogenic phages. Additionally, phages could serve as controllable biosafety switches, and their targeting ability could be engineered into genetic circuits to enable self-destruction of engineered bacteria. Furthermore, in addition to temperature switches, light-controlled switches with lower leakage risks could be used to regulate the release and in vitro suicide of engineered bacteria. However, attention must be paid to gene editing issues during laboratory operations to ensure the viability of strains before administration.

Dr. Ma also mentioned the complementary relationship between the iGEM competition and academic papers: inspiration for projects can be drawn from papers, and paper topics can be derived from iGEM projects, creating a win-win outcome. Integrating the suggestions of Dr. Ma and Huang Yan, a doctoral student in his group, we initially designed a phage prophage induction circuit based on arabinose and temperature sensing. After extensive literature research, we developed a biosafety scheme incorporating phage genes. Whether to conduct experiments on the phage module will be determined based on the specific experimental schedule and biosafety assessments.

Figure 15.Communication between HiZJU-China and Dr. Yingfei Ma with his doctoral student Yan Huang

July 26, 2025-iGEM Hangzhou Meetup & ZJUT-China 2025

At Yuanzheng Qizhen Hotel of Zhejiang University, we participated in an iGEM team project exchange event—"iGEM Hangzhou Meetup" at the invitation of Bio+ Institute of Science & Technology. Ten teams from universities and high schools in Hangzhou and surrounding areas attended the event, with participants including Dr. Dong Shan, Dean of Bio+ Institute of Science & Technology, Mr. Zhang Xiaohan, iGEM 2025 Official Ambassador, and our primary PI Dr. Lian Jiazhang from the College of Chemical and Biological Engineering, Zhejiang University, among others.

During our project presentation, several judges provided valuable suggestions for improvement. For instance, the content of our Human Practices is not yet sufficiently systematic, with gaps in background research and market evaluation, and it needs to be deeply integrated with project iteration. Mr. Zhang Xiaohan also emphasized the distinction between Human Practices and Education, urging us to clearly understand and practice the essence of HP. Additionally, Associate Professor Tan Zhou from Hangzhou Normal University inspired us by noting that semaglutide has severe rebound effects after drug withdrawal, and since its mechanism is well-studied, we could try to research on GLP-1 and GIP dual-target receptor agonists.

At the event, we also learned that many teams are focusing on weight loss, including the next-generation functional oligosaccharide bio-manufacturing platform developed by OTIA Hangzhou from Hangzhou Olive Tree School, and the light-induced GLP-1 and insulin fusion expression project—LEGO designed by WLSA Shanghai Academy. This indicates that numerous teams are currently focusing on obesity, a critical global issue, and that glycemic control and weight loss can be achieved through multiple approaches.

After that, we had in-depth exchanges with ZJUT-China from Zhejiang University of Technology. They suggested that we could pay attention to evaluating bacterial death during hydrogel embedding and carrier preparation, introduce kinetic models of bacterial death, and explore whether bacterial colonization and adhesion can be achieved through modified hydrogel carriers. With a shared background in bioengineering, we have become increasingly aware of the importance of hardware and models in our projects.

This event not only facilitated the sharing of existing project designs and preliminary results but also built a communication bridge for teams in Hangzhou and the surrounding areas, helping us establish a small iGEM community. This in turn promotes the applications and interactions of synthetic biology, providing convenience for the final improvement of our project and the expansion of Human Practices. In the future, we hope there will be more such events to bring iGEMers together and share our boundless creativity.

Figure 16.Members of HiZJU-China at the iGEM Hangzhou Meetup Venue

August 7, 2025-Synbio Challenges 4.0

On August 7, 2025, our team participated in the Synbio Challenges 2025 held in Shenzhen, China, with numerous iGEM teams from around the world. During the event, we presented our project to judges, corporate representatives, and other competing teams, engaging in extensive discussions. This experience not only enhanced our systematic understanding of the current applications of synthetic biology in health, medical and treatment, but also provided us with valuable feedback for optimizing our project.

During the project defense session, the judges raised several questions and suggestions, including considerations regarding potential immune and allergic reactions caused by our engineered product, the emphasis on the unique advantages of the long-lasting in vivo expression approach, and a more detailed explanation of the principles behind the green tea metabolite-responsive elements.

We also engaged in in-depth discussions with other teams during the academic poster session and received valuable feedback. AMU-China from Army Medical University raised questions regarding the potential adverse effects of our GLP-1-based living drugs, drawing a parallel to semaglutide, which is known to cause severe gastrointestinal discomfort and vomiting, and noting that our product may pose similar risks. The team from Sun Yat-sen University expressed concerns about strategies to prevent the loss of recombinant plasmids from engineered strains in the context of an antibiotic-free environment.

Moving forward, we will address these concerns through further literature research or experimental validation, refining our biosafety design accordingly.

Figure 17.Members of HiZJU-China at the Synbio Challenges Venue

March 8, 2025-Prof. Xiangrui Liu

How E. coli is delivered to the gut and colonizes the small intestine is a crucial issue our project must consider. Professor Xiangrui Liu, a young top-tier talent under the "Thousand Talents Program" of Zhejiang Province and a professor at the School of Basic Medical Sciences, Zhejiang University, focuses on nanomedicines, innovative drug formulations, oral absorption of drugs and dietary supplements, and small peptide drug delivery. Fortunately, we proactively sought to consult him on the intestinal delivery of engineered E. coli.

Professor Liu noted that the highlight of our project lies in the proposal of a novel drug delivery mode, "between endogenous and exogenous administration". By leveraging colonizing bacteria to sustainably produce human-derived GLP-1, we could eliminate the need for frequent medication. He further stressed that the project should prioritize genetic engineering of the engineered strain itself. Consequently, we could streamline the design of the oral delivery platform. Specifically, he recommended adopting colonic-targeted or enteric-coated capsules such as cellulose-based capsules to ensure intestinal colonization of the bacteria while protecting them from degradation by gastric acid and small intestinal enzymes. Additionally, he suggested using bacterial powder encapsulation technology to enhance the room-temperature viability of the strain, which would facilitate its storage and transportation. In contrast, he advised against complex formulations such as microcapsules and liposomes.

Besides, regarding the protein modification module, Prof. Liu recommended three targeted modifications to the native GLP-1 sequence: enhancing membrane penetration capability, improving enzyme resistance, and increasing structural stability. These modifications, he emphasized, are critical to ensuring the peptide drug reaches the intestine intactly and maintains long-term stability in vivo. For the project's indication selection, he advised prioritizing obesity or chronic disease management over glycemic control. His rationale was that glycemic control requires precise dosage regulation, whereas the uncontrollable GLP-1 secretion levels of engineered strains could pose safety risks such as unpredictable fluctuations in blood glucose, potentially leading to hypoglycemia.

Incorporating Prof. Liu's advice, we decided to connect a membrane-penetrating peptide to the modified GLP-1 peptide, enabling it to be secreted by EcN and cross the intestinal cells into the capillary and blood circulation. In terms of hardware, we would use a relatively simple formulation that has colonic targeting properties, such as a hydrogel plan.

Figure 18.Communication between HiZJU-China and Prof. Xiangrui Liu

March 17, 2025-Prof. Weijun Tong

The selection of intestinal delivery vectors for engineered bacteria is a core challenge for our team: how to maintain engineered bacteria viability against gastric acid and digestive enzymes while enabling their safe, long-term colonization in the gut. Seeking to address this challenge, we were fortunate to connect with Professor Weijun Tong, Associate Director of the Institute of Biomedical Macromolecules at Zhejiang University, and had a discussion focused on the selection of biological carriers.

Our initially proposed candidate carriers for oral delivery of engineered bacteria included enteric-coated capsules, hydrogels, and microalgae-based delivery systems. After a comprehensive evaluation, we determined that hydrogels exhibited superior biocompatibility, excellent capacity to preserve the viability of encapsulated engineered bacteria, and relatively mature, straightforward preparation protocols, features that better aligned with our project requirements. Consequently, we ultimately selected a sodium alginate-calcium chloride cross-linked hydrogel as the delivery vector. This vector enables precise protection and pH-responsive controlled release of engineered bacteria along the gastrointestinal tract.

During the discussion, Prof. Tong offered critical insights from the perspective of end-user acceptance: as a drug delivery vehicle, Escherichia coli (E. coli) would likely face a higher acceptance barrier compared to chemical drugs. For one, E. coli is commonly used in water quality monitoring, which easily fosters the public's inherent perception of it as "non-pharmaceutical". For another, market acceptance of live bacterial therapeutics remains generally low, even if the strain is a chassis organism with FDA safety certification, dissemination of biosafety knowledge and its principles is still necessary to facilitate the translation of this technology into clinical products and effectively improve patient medication adherence.

Beyond user acceptance, Prof. Tong further noted that even if peptide drugs are successfully released in the intestine, they remain highly susceptible to degradation by intestinal proteases. Thus, in addition to optimizing the in vivo circulation half-life of GLP-1, we must rationally address the degradation challenges of GLP-1 during the post-intestinal release phase.

Prof. Tong emphasized that the project must strengthen the contextualization of "end-user pain points." Only by helping the public clearly identify the limitations of current hypoglycemic treatments such as the inconvenience of injectable administration and side effects associated with chemical drugs can the innovative approach of "intestinal probiotics producing GLP-1" become a more acceptable medication choice.

Figure 19.Communication between HiZJU-China and Prof. Weijun Tong

July 31, 2025-iGEM Westlake 2025

Considering that other universities in Hangzhou participating in iGEM including Westlake University, we specially invited iGEM Westlake 2025, who have a solid foundation in biology, to jointly discuss issues related to project design.

During the discussion, we observed that Westlake also employed hydrogels as their delivery system. We reached a consensus on hydrogels' advantages in biocompatibility, environmental responsiveness, and loading capacity. They further suggested that the hydrogel carrier could be optimized to enhance its in vivo colonization ability if time allowed.

This online exchange not only expanded our view of mammalian cell system experiments and CRISPR-Cas technology, but also highlighted the significance of hardware carriers in live bacterial delivery, which is particularly crucial for our gut probiotic delivery project.

Figure 20.Online communication between HiZJU-China and Westlake

August 12, 2025-Dr. Shiqun Shao

Due to the lack of animal experimental conditions, we planned to build an in vitro intestinal model to characterize the actual effects of the gene circuit. The preliminary plan was to use a continuous culture reactor to simulate the stable microbiome density in the intestine. To assess the feasibility of constructing this physical model, we consulted with Dr. Shiqun Shao, who taught us the specialized course Biochemical Reaction Engineering.

After listening to our intent and initial design of the turbidostat, Researcher Shao considered the proposal to be valuable for exploration. By integrating the review of the biochemical reactor's kinetic model with the knowledge of this model acquired in the course, we further clarified the connection between mathematical modeling and practical hardware construction, and refined the scheme for determining the induction time of the target gene under continuous culture conditions. For the specific implementation, Dr. Shao provided several suggestions: using an oil bath to maintain a constant temperature for the reactor and a peristaltic pump for continuous feeding and drainage; supplying nitrogen gas to the three-neck flask to simulate an anaerobic environment; measuring the μ_max (maximum specific growth rate) and K_s (half-saturation constant) of the target strain under specific growth conditions, based on the growth curve plotted with substrate concentration gradients; using the Monod or Logistic equation to model the growth curve, as well as feasible modification schemes for the preset continuous stirred-tank reactor (CSTR) assumption; introducing mixed bacteria to simulate the real environment of the human gut; and the sterilization conditions for various equipment. These suggestions provided critical support for our hardware construction and modeling derivation.

In addition, Professor Shao generously provided the constant temperature water bath equipment and answered our team's questions regarding the intestinal microbiome simulation modeling, guiding us to apply the knowledge learned in class to solve practical problems.

Figure 21.Communication between HiZJU-China and Dr. Shiqun Shao

September 3, 2025—Prof. Zhuxian Zhou

Given the poor colonization stability of E. coli Nissle 1917 in the intestinal tract, we plan to enhance the intestinal colonization ability of EcN by conducting thiolation modification on hydrogel carriers, which will strengthen their interaction with the intestinal mucosal mucus [11].

To advance this scheme, on the afternoon of September 3, 2025, we consulted Professor Zhuxian Zhou from the College of Chemical and Biological Engineering, Zhejiang University, regarding the technical details related to the thiolation modification of hydrogels. Professor Zhou has long been engaged in the field of intelligent biomedical polymer carrier materials and has achieved fruitful results in the precise synthesis and modification of materials. He also serves as an associate editor of the international journal Chemical Engineering Journal, possessing profound theoretical knowledge and rich practical experience. After fully understanding our research ideas, Professor Zhou first clearly affirmed the feasibility of the thiolation modification scheme, pointing out that the hydrogel carriers modified by thiolation will undergo adaptive changes in their physical and chemical properties, enabling them to form a stronger interaction with the intestinal mucosal mucus and providing key support for improving the colonization ability of the strain.

On this basis, Professor Zhou further optimized and finally confirmed the specific experimental protocol for the "bacteria-embedded thiolated hydrogel carrier" in combination with our research needs, and at the same time encouraged the team to actively carry out subsequent experimental verification. In addition, to address the material and equipment needs during the experiment, Professor Zhou generously provided the key material CMC-SH (thiolated carboxymethyl cellulose) and a freeze dryer, offering great support for the conduct of our dry lab experiments.