Human Practices

Overview

To address the significant public health challenge of zoonotic diseases and promote the safe, sustainable application of antimicrobial peptides (AMPs), we have developed a pet antibacterial spray product. From the outset, we embedded responsible innovation principles into our project design, ensuring that safety, ethical considerations, and stakeholder engagement guided every stage of development.

However, to ensure safety and scientific accuracy, we investigated various official and literature data to prove the project's requirements. Meanwhile, we also conducted online questionnaires to collect data from individual pet owners as the strongly associated target users of the product and invited several experienced pet owners for in-depth conversation to ensure the technical potential of pet care and people's daily needs. Moreover, places where pets are concentrated, such as catteries, are also our communication targets. This helps us understand more about the application scenarios, procurement standards and cooperation intentions of antibacterial sprays for pet antibacterial products in daily operations. Not only that, in order to further understand the problems of the products, we conducted an interview with a pet products operating company. During the experiment, we also sought out Professor Duan, Ms.Cen, Doctor Sun,a veterinarian, and Dr.Li, the chief scientist of pet medicine research and development, for in-depth exchanges on the difficulties encountered and issues related to the development and market launch of pet medicines.

Inspiration

Nowadays, keeping pets has become a common way of life. While pets bring endless joy and companionship to people, they also make people pay more and more attention to their health. In the warm space where pets can be together, preventing diseases with pets has become a key task for families to raise pets. Imagine that the pet at home is lively and cute, but it may inadvertently carry germs and threaten the health of the family, which undoubtedly casts a shadow on the happy life of the pet. Therefore, how to effectively disinfect and sterilise has become the focus of attention for pet-raising families.We have thoroughly investigated the disinfection and sterilisation products currently on the market for families and pets. These products can be roughly divided into the following categories, antibiotics, chemical bactericides, plant essential oils.

Antibiotics are prone to inducing antimicrobial resistance, chemical disinfectants are irritating and can accumulate toxicity, plant essential oils have limited bactericidal efficacy. It is evident that these products cannot simultaneously achieve safety, low irritation, strong bactericidal power, and the absence of antimicrobial resistance.

In contrast, antimicrobial peptides offer advantages such as broad-spectrum activity, high safety, no induction of resistance, and environmental friendliness, demonstrating great potential as antibacterial and protective products for both humans and pets.[2]It also meets people's expectations for products that are both effective and safe.

1. Background Research：The Increasing Risks Coming with the Increasing Numbers

With the development of human society, the concept of pets as "family members" and "friends" has become increasingly ingrained. Although China's pet industry started later compared to European and American countries, it has developed rapidly over the past 30 years. According to the "2024 China Pet Industry Research Report" released by iResearch and the "China Pet Industry White Paper 2024," the number of urban pets in China has been rising year by year in recent years, with dogs and cats being the primary types. By 2024, the total number had reached 124.11 million. The pet consumption market size has also maintained steady growth.

Concurrently, expectations regarding the daily care of pets have steadily increased. Recognition has grown that certain microbes are zoonotic pathogens, such as Staphylococcus aureus and Salmonella. Disinfection and sterilisation to prevent the transmission of pathogens between pets and humans have consequently become integral to routine preventative measures. However, the selection of disinfectant products, alongside concerns regarding their efficacy and safety, has emerged as a growing point of consideration for pet owners.

2. Multi-dimensiona Survey Encompassing Users,Markets and Professionals

1） The Needs of Public and Pet Owners

a) Public Questionnaire Survey: Basic Understanding of User Perception

We distributed online questionnaires to understand the pet-keeping trends among people around us and their views on zoonotic diseases and pet antibacterial products. The results show that people have a certain understanding of zoonotic diseases, show concern about bacterial transmission, have a significant practical need for preventing and dealing with related diseases, and have a relatively high acceptance of antibacterial products. All these have laid a market foundation for the promotion of new antibacterial products.

The following is a detailed analysis:

Among the 314 collected questionnaires, 283 respondents are currently keeping pets or plan to do so. Among these 283 respondents, 87% reported having "heard of zoonotic diseases," and 79% expressed being "Worry about bacterial transmission between humans and pets."

These figures indicate that current and prospective pet owners have a certain level of awareness regarding zoonotic diseases.

53% reported "regularly performing antibacterial or disinfection treatments on pet supplies/environments," and 30% indicated that "they or their relatives/friends have previously contracted bacterial-related diseases." These relatively high percentages suggest that preventing and addressing zoonotic diseases has become a practical need worthy of attention.

Meanwhile, in response to the question "Have you or someone close to you ever contracted a bacterial-related disease?", the option "unclear" received the highest proportion of responses, indicating that a considerable number of people still lack accurate or deep awareness of the existence of such pathogens and diseases.

This made us acutely aware of the importance of popular science education, so we also planed to arrange targeted promotional activities accordingly. (Please refer to 5 for the detailed introduction of this live-streaming science popularization activity.)

Regarding antibacterial products, the vast majority of respondents reported using existing products on the market, such as alcohol-based spray and hypochlorous acid solution, with only 8% indicating they have never used any antibacterial product. In terms of willingness to "try" antimicrobial peptide products, 48% of respondents expressed willingness. This indicates a high level of acceptance of antibacterial products among pet owners, laying a solid market foundation for the promotion of novel antibacterial products.

b) Pet Owners' Discussion: Key Concerns and Product Expectations

In order to further understand pet owners' opinions on existing disinfection and sterilization products, as well as their demands and expectations for new antibacterial products, we organized a symposium. Five senior pet owners were invited. They are teachers from Ulink College of Suzhou Industrial Park, each having multiple pet cats and dogs at home, and all with over five years of pet-raising experience. Also, we hope to learn about the current situation of antibacterial measures for pet owners' home environments and their pets' body surfaces.

The teachers indicated that pet owners generally have basic knowledge of zoonotic diseases and have, to some extent, encountered zoonotic pathogens—such as ringworm—in their experience. They noted that once either a pet or a human contracts such a disease, the treatment process is very troublesome. In hopes of a quicker recovery, people often resort to "home remedies." Moreover, they emphasized that prevention is more important than treatment.

When discussing daily antibacterial practices and choices of antibacterial products, the teachers unanimously stated that while they pay attention to hygiene and cleaning frequency in daily care, they rarely perform dedicated antibacterial treatments. Upon further inquiry, we learned that their lack of trust in commercially available pet-specific disinfectants is a key reason. They either distrust these products or believe there are no genuinely effective and safe options specifically designed for pets. As a result, they prefer household disinfectants—such as chlorine-based solutions to clean pet supplies or alcohol sprays to disinfect their own skin surfaces.

Regarding expectations and concerns about new products, all teachers agreed that "safety and harmlessness to pets" is the most important factor—this is one of the reasons we chose antimicrobial peptides.

They also highlighted pain points with current antibacterial products:

• most existing antibacterial and disinfecting products on the market do not clearly indicate whether they are safe for pets, making it difficult to assess potential risks;

• the authenticity and credibility of product claims are hard to verify, and actual effectiveness is questionable;

• commercial pet cleaning and antibacterial products often fail to adequately cover real-life pet-owning scenarios.

Regarding the new antimicrobial peptide spray,

• some teachers expressed concerns about its production using E. coli expression systems. Providing safety testing reports would help improve acceptance.

• They hope the spray would be fast-drying, have a pleasant scent, offer strong antibacterial efficacy, and be safe for children. They think the spray is the most convenient.

• They hope the application scenarios can be more extensive, with the pet's body and the area around the pet toilet being the key usage areas.

• In terms of price, we can refer to the price of conventional disinfection products on the market, and the premium of about 20% is acceptable, but whether consumers will continue to buy it ultimately depends on the actual effect of the product.

In terms of product optimization and usage scenarios, the teachers identified pets' bodies and areas around pet toilets as key application zones. One teacher mentioned that if the product could help special households—such as those with newborns or pregnant women—reduce concerns about zoonotic pathogens, it would be highly valuable. They also believed that sprays are the most convenient format for home use, though wipe-type products are also worth considering.

2）The Needs of Market

These interview with catteries of various sizes and pet product companies aims to gain an in-depth understanding of bacterial infections affecting both pets and humans within pet service facilities, and explore the unmet market demands of current pet antibacterial products.

From the viewpoints of Ms. X, Mr. Kaer, and Ms. Yang, we can observe some common market issues regarding the products, namely safety and non - irritation, easy storage, large - scale use, and cost control.The following is a detailed analysis.

a）Interview with Catteries: Special Demands in Large-scale Scenarios

We communicated with a cattery owner who keeps 200 cats and offers various pet-related services.

She mentioned that bacteria commonly infecting cats in her cattery include Salmonella and E. coli. To prevent illnesses caused by pathogens, she regularly uses antibacterial or disinfectant products to sanitize the environment. However, this inevitably causes issues such as irritation, and even stress-related behaviors in the cats.

Regarding antibacterial products with antimicrobial peptide, she described them as "relatively safe," "water-soluble," and "gentle." However, she expressed concerns about their "higher price" and potential ineffectiveness against certain bacteria. In addition, she emphasized that such products must undergo safety testing and be used strictly according to the instructions.

Mr. Kaer stated that Malassezia and ringworm are typical zoonotic becteria/diseases shared between humans and cats, and bacterial infections such as coccus also frequently trouble him. Currently, his cattery is managed through zoning, isolation, and daily cleaning to minimize the spread of bacterial contamination.

Regarding expectations for antibacterial products, he said, "Most disinfectants currently used in pet environments still require preventing pets from licking them. It would be better if there were a product with extremely high safety for pets." Therefore, upon learning that antimicrobial peptide products combine high safety with broad-spectrum antibacterial properties, he expressed strong interest and anticipation, firmly believing that such products have the potential to address the current pain points in practice.

As for product packaging design, Mr. Kaer hopes it can be suitable for large-scale cleaning and disinfection. For example, effervescent tablets are not only convenient for storage and transportation but also allow flexible adjustment of concentration based on actual needs. At the same time, in terms of scent, irritating ingredients such as fragrances should be avoided as much as possible, opting instead for mild, odorless formulations to reduce olfactory irritation for cats.

In addition, Mr. Kaer told us that many members of the public still hold strong reservations toward cats and have limited understanding of pet-related health issues. Therefore, he believes that alongside promoting the product, carrying out relevant science communication efforts is a long-term and important mission. He hopes to leverage professional expertise to guide the public and help people correctly understand knowledge about pet care and health protection.

Regarding the storage issue,we also interviewed Dr. Li, he raised the storage issue of the product--how the product can be stored for a maximum period?So we then met professor Duan to obtain several crucial advises, she admitted the difficulty of keeping the solution, which is one of the key issues we have to figure out in the competition. Professor Duan suggested that we can produce the antibacterial peptide in freeze-dried powder for storage, extending the best-before date of out product.

b）Pet Products Operating Company: User Demands and Product Optimization Directions

To learn about more problems of the product in the market, we connected to Ms. Yang, who is currently taking in charge of products relating to pets. During our communication, she has pointed out, throughout the consultation with the pet owners, that whether the product has aroma or not and the level of safety to pets are the most striking issues which customers are concerning about. For aroma, it should better be completely avoided, preventing pets from getting disturbed.

Regarding the packaging design of the product, Ms. Yang said that customers prefer to use sprays as this form of product is convenient to use. If a divided-packaging design is adopted, efforts should be made to minimize the trouble caused by proportioning for customers.

3) Veterinarians and veterinary drug professionals: AMPs have great application potential

After learning about the expectations of users and the market for the new product, we further interviewed pet doctors and veterinary drug R & D experts. We hoped to examine the application potential of antimicrobial peptides as a new type of pet antibacterial product from the professional perspectives of veterinarians and veterinary medicine. Ms. Cen, Dr. Sun, and Dr. Li all said that antimicrobial peptides have great potential. However, Dr. Li indicated that there are still many difficulties to overcome on the way to the real market.

a) The Application Potential of Antimicrobial Peptides

Ms. Cen mentioned that during daily diagnosis and treatment, bacterial or fungal infections in pets are quite common, accounting for approximately 20% of all clinical cases and gradually becoming one of the major issues affecting pet health. Antimicrobial peptide products, which don't induce drug resistance, can perfectly compensate for the drawbacks of traditional antibiotics. From the perspective of clinical application, these products can not only effectively combat common bacterial or fungal infections in pets but also avoid the risk of drug resistance, meeting pet owners' needs for safe and efficient treatment solutions. Therefore, they are likely to be widely welcomed in clinical practice.

Doctor Sun told us that in her daily work, she often comes across cases where the disinfectants bought by pet owners are highly irritating or toxic to pets, resulting in pet skin infections or illnesses. Also, some pet owners don't use antibacterial products as per the doctor's instructions when treating pet wounds, leading to repeated wound infections. Therefore, she believes that in addition to having certain disinfection and sterilization capabilities, new antibacterial products also need to identify the target group and carry out targeted promotion. Moreover, these products should have the potential to comprehensively meet the health needs of pets.

b) Potential and Challenges in Entering the Market

Dr.Li believes，antimicrobial peptides, as a new biological agent, is becoming one of the important directions of veterinary drug research and development because of its broad-spectrum antibacterial properties, resistance and high safety.As Dr. Li pointed out in the interview, "the veterinary drug industry has been exploring milder, safer, and low drug resistance. The production cost and biological activity in the preparation process are the key to whether the product can go to the market.

In addition, in terms of the management and approval of veterinary drugs, Dr. Li said, "at present, there are no antibacterial peptides used for pets or non oral animal products in China, so these products may be under the management of the Ministry of agriculture for the time being." Dr. Li emphasized that "it is strongly recommended to intervene in the registration path planning and regulatory consultation at the early stage of R & D, which is equivalent to strategic planning."

Although Dr. Li believes that currently, antimicrobial peptide products do not show an advantage in terms of price, they still have good development potential in the veterinary drug field, especially in the pet health market.

3. Design and Screening of the Antimicrobial Peptides

Combining the public's demand for'safety and non-irritation', the catteries' expectation for 'broad-spectrum antibacterial properties', and the 'production pain points' pointed out by Dr. Li, we carried out technical optimization on the activity, toxicity, and stability of antimicrobial peptides. The specific plan is as follows:

a) Prof. Duan：

To address these issues and optimize our experimental approach, we consulted Professor Lengxin Duan, an expert in synthetic biology. Below is a summary of her key suggestions and the subsequent refinements made to our experimental design.

Improving AMP Activity and Optimizing Expression

To tackle the issues of low stability and activity of short AMPs, we initially planned to use point mutations to enhance their performance. Prof. Duan emphasized the importance of clarifying fundamental properties of the peptide—such as the number of amino acids, primary structure, and expression form (soluble or inclusion body). For shorter peptides, she suggested considering an exosome-based expression system as a more efficient and cost-effective alternative to traditional mutagenesis libraries. She also highlighted the necessity of identifying the unique advantages of our AMPs compared to existing drugs, including antimicrobial spectrum and potential for low resistance development.

Reducing the Impact of AMPs on Host Cells

To mitigate the toxicity of AMPs to E. coli host cells, we proposed a co-expression strategy with anionic antioxidant peptides. Prof. Duan supported this approach and further recommended incorporating resistance genes via a dual-plasmid system to enhance host robustness. Careful consideration of compatibility between genetic elements is essential for success.

Validation of Antimicrobial Efficacy Under Safety Guidelines

Due to iGEM safety regulations, we were unable to use pathogenic bacteria such as Staphylococcus aureus directly. Instead, we adopted two alternative strategies:
a) Molecular docking simulations to predict binding affinity between AMPs and target pathogens;
b) Bacteriostatic assays using non-pathogenic model strains (E. coli and B. subtilis) to infer efficacy against Gram-negative and Gram-positive pathogens.

Prof. Duan recommended combining both methods to strengthen validity—experimental results offer direct evidence, while simulations provide mechanistic support. She specifically suggested focusing docking studies on common pet-related pathogens to improve relevance.

Formulation and Safety Assessment

For the final product formulation, Prof. Duan advised using lyophilized powder rather than liquid solution, as it significantly improves AMP stability and shelf life. Key challenges during preparation include controlling peptide degradation, maintaining sterility, and optimizing effective concentrations (e.g., MIC, IC₅₀). If peptides are short enough, chemical synthesis may reduce production costs compared to fermentation.

Regarding safety, she emphasized that systemic assessments—including cytotoxicity, hemolysis, and animal testing—are essential. Although such tests are beyond our current scope, outlining a clear safety validation plan is critical for future development.

Conclusion

Through this consultation, we identified key refinements in AMP design, expression, validation, and translational planning. Prof. Duan’s insights not only helped optimize our experimental design but also provided a clearer pathway for project development. While challenges remain, these recommendations have greatly enhanced the scientific rigor and feasibility of our approach.

b) Dr.Li：

After we introduced the project background and technical route to Dr. Li in detail, he put forward professional opinions on the strategy of using E. coli expression system to produce antimicrobial peptides, and pointed out that there were still many key challenges in the technical feasibility, production process and industrialization of this path.

Difficulty 1: Antimicrobial peptides have antibacterial or toxic effects on the host bacteria - Escherichia coli itself.

Antimicrobial peptides have antibacterial or toxic effects on the host bacteria - Escherichia coli itself. Antimicrobial peptides mainly play a role by destroying the cell membrane structure, and as a prokaryotic expression system, the cell membrane structure of Escherichia coli is also the target of antimicrobial peptides. Therefore, in the process of expression, antimicrobial peptides may directly affect the growth state and viability of host bacteria, resulting in low cell density, prolonged fermentation cycle, and even the collapse of expression system. This puts forward extremely high requirements for the construction of engineering strains. It may be necessary to reduce toxicity by optimizing promoters, using inducible expression systems, or designing secretion expression of antimicrobial peptide sequences, but this also increases the complexity and uncertainty of the system.

Difficulty2：E.coli contains a variety of endogenous proteases, which can recognize and degrade exogenous or abnormally folded polypeptide molecules.

Therefore, even if the expression of antimicrobial peptides can be achieved, its actual yield may be greatly reduced due to degradation. In order to improve the stability, it may be necessary to introduce molecular chaperone coexpression, protease deficient strain, or antimicrobial peptide sequence for codon optimization and structural modification, but whether these strategies will affect its final activity still needs a lot of experimental verification.

Difficulty3：The correct folding of antimicrobial peptides is as important as post-translational modification.

Many antimicrobial peptides need to form specific spatial structures to exert biological functions, while Escherichia coli lacks eukaryotic specific modification mechanisms (such as some glycosylation or lipidation processes), which may lead to low product activity. In addition, if there are disulfide bonds and other structures in the antibacterial peptide molecule, its correct pairing and stability are also the quality control points that must be controlled in industrial production.

Difficulty4：The difficulty of developing the downstream separation and purification process.

Without an efficient, low consumption and scalable purification scheme, the purity, activity and batch consistency of the final product will be difficult to meet the requirements of veterinary drug registration, let alone realize large-scale production.

In conclusion, although the production of antimicrobial peptides using E. coli expression system has certain advantages in cost and scale, its technical challenges cannot be underestimated. From gene construction, strain optimization, fermentation process to purification strategy, each link needs systematic research and careful pilot scale-up verification. Only by making breakthroughs in these key technology nodes can we lay a solid and reliable foundation for the final industrialization and marketization of such products.

Technical Solutions：

Following Prof.Duan and Dr. Li's advice, we have taken further action.In order to deal with the two main problems encountered in the production of antimicrobial peptides - toxicity to Escherichia coli and easy degradation - our team plans to take some milder and direct improvement methods.

1.Make antimicrobial peptides lose their charge and reduce toxicity

The reason why antimicrobial peptides are toxic to host Escherichia coli is that its mechanism of action is to destroy the bacterial cell membrane. After binding, the antimicrobial peptides will insert into the cell membrane and aggregate to form pores, resulting in the leakage of cell contents and the rupture and death of the bacteria.

When we transfer the exogenous antimicrobial peptide gene into E. coli to make it abundantly expressed, these antimicrobial peptides will also target the cell membrane of the host bacteria, thus inhibiting the growth of the bacteria and even causing the death of the host cells, seriously affecting the fermentation yield.

We thought of a way: temporarily match it with a negatively charged peptide, that is, an anionic peptide, and let the two combine into a neutralizing fusion protein. In this way, the overall charge tends to balance, and the antimicrobial peptides cannot recognize and attack the cell membrane of the host bacteria, thus greatly reducing the toxicity.

We will set a switch between the antimicrobial peptide and this peptide, which is a sequence that can be recognized and cut by a specific protease. After the fermentation is completed and the protein is purified, we add this protease in vitro to accurately cut off the anionic peptide and release the active antibacterial peptide, so as to reduce the toxicity of antibacterial peptide to host cells.

2.Multi step protection to prevent antimicrobial peptides from being degraded

We intend to minimize this problem by adding protective agents in the purification process, and appropriately add some protease inhibitors to protect the antimicrobial peptide from degradation by residual enzymes in the cell fragmentation and subsequent purification stages. These additives will be completely removed in the final product to ensure safety.

Summary

The core idea of this scheme is to "protect first and then release" -- let the antimicrobial peptide be expressed safely first, and then restore its activity after purification. Although this method will add some steps in operation, such as one more step of enzymatic digestion and purification, it can effectively solve the bottleneck problem of toxicity and stability.

We believe that these intuitive and easy to implement methods can effectively improve the production efficiency of antimicrobial peptides and lay a solid foundation for the development of antimicrobial spray products for pets. Next, we will gradually test and optimize each link to ensure the effectiveness and safety of the final product.

4. Product Design and Safety Verification

a)The Broad-Spectrum Verification

Through bioinformatics methods and analogical inference, we used the amino acid sequence database of the target antimicrobial peptide and the surface protein structure data of common pet pathogenic strains to conduct molecular docking simulations, so as to predict its binding ability to different pathogenic bacteria. Meanwhile, by analogy with the inhibitory spectrum of the reported homologous antimicrobial peptides against a variety of microorganisms, we preliminarily inferred that the antimicrobial peptide in this study may have broad-spectrum inhibitory potential against Gram-positive bacteria, Gram-negative bacteria and some fungi, which provides direction and theoretical basis for the selection of strains in subsequent in vitro bacteriostatic experiments.

b)The Safety Verification

Research has found that derivatives of the antimicrobial peptide Granulysin can cause potassium ions (K+) to leak out of red blood cells, leading to continuous uptake of sodium ions (Na+) from the outside. This results in increased concentrations of Na+, Cl-, and Ca2+ inside the cell and a decrease in K+ concentration, causing passive water transport into the cell, which disrupts the stability of red blood cells and ultimately leads to cell lysis. To reduce the risk of hemolysis in mammals, we have lowered the minimum inhibitory concentration (MIC) of the antimicrobial peptide and collected MIC data, while also consulting relevant international regulations and guidelines to ensure that the hemolytic activity of the peptide at this concentration is below the safety threshold. In subsequent product purification steps, we will consider removing the residual toxicity of E. coli.

c)The Target User and usage scenarios of Product

Combining the early - stage market research and user demand analysis, we focus on three core target user groups. The first is the middle - and high - end urban pet owners who pursue a high - quality pet - raising lifestyle and pay attention to product safety and user experience. The second is the families where children and pets co - exist. They have a more urgent need for antibacterial and non - irritating pet environments, as they need to take into account the health of both children and pets. The third is the B - end scenarios such as cat cafes and pet clinics. In these scenarios, the pet turnover rate is high and the antibacterial demand frequency is also high. There are clear requirements for product cost - performance and practicality.

In response to the differences in usage scenarios and demands of different user groups, we have also designed the product packaging accordingly. Please refer to the following d) Hardware—Product Packaging Design.

d)Hardware—Product Packaging Design

At the beginning, we intended to design a spray bottle because it is the most convenient to use. However, considering the instability of antibacterial peptides when stored in solution, after talking with Professor Duan and inspired by some independently sealed beverage packaging on the market, we designed a dual - chamber spray bottle with a dry - wet separation feature.

Honey is sometimes sealed separately and only mixed with the drink immediately before consumption.

The Dual-Chamber Bottle（150mL Household Package）

To meet the demand for "safety, non-irritation, and portable use" in families with children or those with a small number of pets, we designed a 150 milliliter dual-chamber spray bottle. The specific structure is as follows:

Our unique dry-wet separation spray bottle finally comes out to be the best fitting solution. Inside, the core technique is producing two distinct chambers working in harmony:

Small Chamber (Dry): With space in vacuum, securely holds the freeze-dried AMP powder, which is sealed with a thin aluminium membrane.

Large Chamber (Wet): Holding 150mL of the solvent ready to dissolve the powder.

After pressing the nozzle, a plunger mechanism connected to it will simultaneously move downwards. Several sharp piercing tips attached to this plunger will then puncture the aluminium membrane at the bottom of the small chamber, allowing the freeze-dried AMP powder to fall freely into the large solvent chamber below.

In addition, we designed a one-way valve mechanism linked to a separate syringe switch at the bottle neck. After the initial mixing is completed, if residual powder remains in the dry chamber, the user can press the one-way valve switch to allow solvent to flow back into the upper chamber, enabling a secondary dissolution. This prevents incomplete dissolution of the freeze-dried powder, which could otherwise result in lower peptide concentrations and reduced antibacterial efficacy.

Usage scenarios: For families with certain usage habits and demands. They conduct disinfection and sterilization no more than 3 times a day, with each use of about 5-10 milliliters. This product can last for about two weeks(within the time of the stability verification experiment), ensuring both effectiveness and avoiding the need for frequent restocking.

Small-Dose Packaging（10mL Portable Package）

Considering the public's concern about the effectiveness of the new product, based on the design of the large-capacity spray bottle, we further optimized the hardware structure and developed a 10-milliliter small-package spray bottle. It can be used not only as a trial pack but also for outdoor use.

As shown in the figure, the bottle body is more compact, yet retains the core concept of "dry-wet separation": The freeze-dried powder is pre-stored in the upper micro-chamber and sealed with a breakable film. The lower chamber contains a single-use dose of solvent.

Compared with the large-capacity spray bottle, the small-packaged design eliminates the risks associated with repeated opening and long-term storage. Each unit is designed for single-use or short-term use, effectively reducing the possibility of peptide inactivation.

Usage scenarios:It is designed for families with small - volume demands. They can open it at any time, and won't feel bad even if they discard it after expiration. It's also convenient for outdoor use, such as when taking pets out or traveling.

The transition from the dual - chamber design to the addition of small - capacity spray bottles reflects our optimization in hardware design, making the antibacterial spray more in line with users' actual needs. This is also a crucial step in the transformation of the product from laboratory research to daily-life applications.

B-end Combination Set（500mL+10 pieces 10mL Trail Packs）

To meet the catteries' demands for "large-scale disinfection" and "cost control", and take into account the bulk use and customer experience in B - end scenarios, we also plan to launch a combination set. The 500-milliliter large-capacity spray bottle is suitable for daily whole - store disinfection in catteries, pet stores, and clinics. The 10-milliliter one can be used for customers to experience or for temporary outdoor use. By verifying the effectiveness first before making a purchase decision, we can address users' concerns about "effectiveness".

Both capacity spray bottles can continue to adopt the dual - chamber structure with dry - wet separation.

Key Features & Advantages

Maximum Potency Preservation: By storing the AMP as a freeze-dried powder completely separate from the solvent and under vacuum, we drastically minimize degradation, ensuring peak antimicrobial activity at the time of use.

Effortless Activation & Mixing: Activation is intuitive – simply press the nozzle once to initiate the mixing process. Users then gently shake the bottle for a few seconds to dissolve the powder completely, creating a ready-to-use solution.The one-way valve mechanism can better ensure that there is no residue of the freeze - dried powder.

Guaranteed Precise Mixing: The design ensures the entire powder dose reliably enters the solvent chamber, guaranteeing the correct concentration every time.

Convenient Spray Application: The final solution is delivered via a standard, easy-to-use spray nozzle, allowing for targeted and mess-free application onto pets.

Enhanced Stability & Shelf-Life: The vacuum-sealed dry chamber provides superior long-term stability compared to pre-mixed liquid solutions.

Our intelligently engineered dual-chamber spray bottle is more than just packaging; it's the key to unlocking the full potential of Antimicrobial Peptides for pet care.

The dry-wet separation and vacuum-sealing address the key "easy-storage" problem raised by Mr. Kaer and Dr. Li, while the combined design of the two chambers and the one-press operation solve the "user-friendly" issue mentioned by Ms.Yang and the teachers in the discussion.

This innovative design embodies our iGEM team's commitment to applying synthetic biology principles to create practical, real-world solutions for animal health and well-being.

5. Science Popularization Education and User Trust - building Plan

Background of the Livestream

Through analysis of the aforementioned online questionnaire and focus group findings, we observe that pet owners exhibit a mature awareness and demand for antimicrobial solutions concerning pets, humans, and household environments. Their concerns extend beyond product safety to include apprehensions about emerging technologies. Concurrently, market regulations require ongoing refinement to restore public confidence in the sector.

To enhance public understanding of antimicrobial peptides and foster accurate perceptions regarding biotechnology safety, we plan to increase external communication opportunities. Given the familiarity and acceptance among today's younger pet owners with online and livestream formats, we have planned a series of collaborative livestream events——Circle of 'LIVE’.

2) Activity Implementation："Circle of 'LIVE’"——Wellness

We collaborated with multiple teams to organize a series of live streaming under the theme "Circle of 'LIVE’".

There were two sessions in this series of live streaming . The topic of the first session was "Contribution", and the second session was "Wellness". Centering around "Circle of 'LIVE", the two live streamings introduced how the projects of each team brought significance to people and society.

Our team's introducation was in the second one, with the topic of wellness, which means ensuring a happy life for people. This aligns with our project's goal of adding an extra layer of insurance for the health of pet owners and their pets. At the beginning of the live stream, we played a promotional video highlighting the current threat of zoonotic diseases and introducing the basic concepts of our project along with the simple working principles of antimicrobial peptides.

Afterwards, we used a presentation to promote from three aspects: popularizing the basic concepts of synthetic biology, introducing the background of zoonotic diseases, and showcasing our antimicrobial peptide products and market potential. We explained in simple terms that synthetic biology is an interdisciplinary field that integrates biology, engineering, and computer science. It involves rewriting or reprogramming DNA.

We set up an interactive and Q&A session. During interactions with the audience, we found that some viewers were curious about the connections between the research topics of our collaborating teams, while others showed a strong desire to share their thoughts on pet-related topics.

3）Activity Reflection and Future Plan

The number of viewers in this live-streaming reached 535, and the interaction rate was relatively high. The questions from the audience mainly focused on core pain points such as "the stability of antimicrobial peptides", "safety", and "technical costs", which were highly consistent with the findings of our research.

Although this live-streaming activity received good feedback, its long-term impact on people is relatively weak. According to Mr.Kaer's description, the public's fear of pets and the germs they carry cannot be alleviated by just one live-streaming event. Therefore, we need to do more in-depth follow-up science popularization and education work. We plan to deepen science popularization in two ways: ① Produce short videos on "antimicrobial peptide knowledge" (such as "How to judge whether an antibacterial product is suitable for pets") and spread them on Xiaohongshu and Bilibili; ② Collaborate with community pet clinics to carry out offline lectures on "Prevention of zoonotic diseases" and provide free consultations for pet-keeping families.

6. Summary

During our human practice journey, we have not only listened to the voices of pet owners, but also communicated with people in the pet industry.

We have learned about the current pain points of zoonoses in humans and pets, as well as the concerns of the pet industry regarding the safety of existing products and the demand for highly efficient and convenient antibacterial sprays. In addition, through conversations with professionals, we also learned how to improve our overall experiments, such as antibacterial experiments, and more details of pet product development, such as safety work and registration planning. These have enabled us to have a deeper understanding of the user pain points and industry responsibilities behind the products, laying a solid foundation for the subsequent optimization of the products and the advancement of popular science work.

References

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