This project leverages Lactobacillus rhamnosus as a chassis organism to develop a novel solution for the real-time detection and degradation of aflatoxins in dairy products. Lactobacillus rhamnosus inherently has the ability to physically adsorb up to 80% of aflatoxin B1 in the environment, but this binding is not stable. Therefore, we employed genetic engineering techniques to introduce the CotAgold laccase gene, derived from Bacillus subtilis and proven to effectively degrade aflatoxin B1, to enhance the degradation efficiency of aflatoxin B1 [1]. To finely regulate the expression of laccase, we designed a comprehensive regulatory system, including key components such as the T7 promoter, nanobody Nb26, Gaussia luciferase, and the photosensitive protein VVD. This system activates luciferase to produce blue fluorescence in the presence of aflatoxins, which in turn activates T7 RNA polymerase, initiating the expression of laccase.

Furthermore, to prevent the engineered bacterial strains from escaping into the environment and causing pollution, we designed a suicide system based on changes in glucose concentration. This system utilizes the PGlu promoter to control the expression of the suicide gene mazF, which encodes an endoribonuclease that initiates when glucose levels are low, leading to bacterial death without lysis, thus preventing the escape of contents.

This technology not only reduces the risk of aflatoxin B1 in feed and the gastrointestinal tract of livestock but also provides a new solution for the field of food safety. Through this innovative biotechnology, we can achieve efficient detection and degradation of aflatoxins while ensuring the safety of the engineered bacterial strains and preventing their potential environmental contamination. The application prospects of this technology are broad, as it can be applied not only in agriculture and animal husbandry but also extended to other food industries for the detection and degradation of other types of toxins.

Our project is dedicated to addressing the detrimental effects of Aflatoxin on human health through the innovative application of synthetic biology. We aim to develop novel biological methods for the degradation of Aflatoxin, a potent mycotoxin that poses significant health risks to susceptible populations. To achieve this, we have initiated a multi-faceted approach that includes:

1. Conducting extensive market research and interviews to gather public opinion on Aflatoxin degradation. This qualitative data serves as the foundation for our preliminary experimental designs and product development strategies.

2. Engaging with experts in the field to obtain valuable insights and feedback, which are instrumental in refining our experimental protocols and enhancing the feasibility of our project.

3. Focusing on market needs and real-life issues, we strive to create solutions that are not only innovative but also practical and aligned with the pressing demands of society.

4. Our ultimate goal is to mitigate the urgent needs of the public and keep humans away from the damage caused by aflatoxin. Through our project, we aspire to make a significant impact on public health and contribute to the broader effort of reducing the harmful effects of this pervasive toxin.

By combining cutting-edge biological research with a deep understanding of market dynamics and public health concerns, our project is poised to offer transformative solutions in the fight against Aflatoxin contamination.

Aflatoxin contamination is a pervasive issue in animal feed, affecting not only the healthy growth of livestock and poultry but also posing a threat to consumer health through the food chain. The exploration and implementation of rational and effective preventive and control measures have garnered significant global attention.

According to market research conducted, existing technologies for the removal of aflatoxins primarily rely on physical and chemical methods.

Common methods for treating aflatoxins in feed on the market include:

A substantial amount of research and application has been conducted on chemical detoxification methods for aflatoxins. The main prevention and control methods include alkali-ammonia treatment, oxidizing agent treatment, and antimicrobial agent treatment. Alkali-ammonia treatment is suitable for green and silage feed with high moisture content, as well as liquid grain and oil, with high detoxification efficiency. Weak alkali and high-temperature treatment for 60 minutes can achieve an aflatoxin destruction rate of 84.5% in peanut meal. Additionally, methods such as fumigating with litsea cubeba oil and adding appropriate amounts of propionic acid and its salts as mold inhibitors can also remove the toxicity of aflatoxins. However, chemical methods can often lead to secondary contamination of livestock feed and still pose safety issues.

Physical removal methods for aflatoxins mainly include washing, adsorption, high-temperature pyrolysis, and radiation degradation methods. The majority of aflatoxin contamination in grain raw materials is concentrated on the surface of the seeds, and washing with water can remove about 80% of the contaminated toxins. Heating treatment can reduce the levels of aflatoxins, with increased detoxification effects as the heating temperature rises and the heating time extends.

These methods are effective to some extent, but they have limitations, potentially leading to the loss of essential nutrients in food or leaving behind potentially harmful reagent residues.

Synthetic biology offers a promising avenue for developing such innovative solutions. By designing and engineering microorganisms or enzymes to specifically target and degrade Aflatoxin, it is possible to create a more efficient and safe method for Aflatoxin removal. This approach has the potential to be more specific, leaving behind no harmful residues and preserving the nutritional value of the food.

The project's commitment to leveraging synthetic biology for Aflatoxin degradation is well-aligned with the market need and has the potential to make a significant impact on food safety and public health. By filling the gap in the market, the project could lead to the development of a new technology that is not only effective but also safe and sustainable, meeting the urgent needs of the market and consumers.

The project is dedicated to the degradation of Aflatoxin B1 through synthetic biology, which aligns perfectly with market demands and has the potential to fill a market gap. The application of this innovative concept and technology may positively impact food safety and public health. Ensuring safety and sustainable development, it meets the urgent needs of the market and consumers.

The future of Aflatoxin degradation technology is bright, especially with the advent of synthetic biology methods, which are expected to revolutionize the way we tackle food safety challenges like Aflatoxin contamination.

We divided the issues into groups based on synthetic biology and those related to it, and designed questions for each group (public perception of aflatoxin, views on synthetic biology, product design suggestions, etc.). This public feedback helps us better position our product features and address the concerns of the public.

We collected survey information from 103 women and 70 men, most of whom have undergraduate to postgraduate degrees and are generally aware of aflatoxin poisoning incidents around them. They recognize that synthetic biology can promote the degradation of aflatoxins, such as the distribution of aflatoxin in liver mitochondria (50%), which can reduce the enzymatic activity of liver mitochondria, cause necrosis of liver cells, and prevent poisoning, such as adding degradable drugs during processing to block the source. It is reassuring that the public believes that synthetic biology degradation methods have advantages, including innovation, harmlessness, and preventability. The public is generally concerned whether synthetic biology technology is absolutely safe, worried that the use of engineered probiotics or gene-editing techniques may lead to unknown risks or the production of new harmful substances, and side effects such as allergic reactions. At the same time, the public is concerned about how to deal with the degradation of engineered bacteria and the nutritional value and taste of food treated with synthetic biology technology. More people express support for the application of engineered bacteria in our project, while a few are resistant, indicating a reluctance to accept genetically engineered products. This suggests that there are still many aspects of future projects that need to be explored, and some common issues that need to be cared about.

To gain deeper insights into the industry and receive expert guidance, the student team from Beijing No. 5 High School, participating in the International Genetically Engineered Machine Competition (iGEM), held an in-depth consultation with leading specialists from DSM China. This exchange not only provided valuable industry insights for our project but also offered critical guidance for our future research efforts.

During the exchange, DSM experts introduced us to various microorganisms and their metabolic by-products that can contaminate dairy products, emphasizing the potential threats these contaminants pose to product quality and consumer health. The data and case studies they shared made us realize the crucial importance of controlling microbial contamination to ensure the stability of the dairy supply chain.

Focusing on aflatoxin, a specific contaminant of interest to our team, the experts elaborated on the preventive measures implemented across different stages of the dairy supply chain. From strict feed control to the use of safe additives (such as montmorillonite powder and diatomaceous earth) during livestock rearing, and stringent post-production quality testing, the dairy industry demonstrates its commitment to food safety. Additionally, the experts discussed the commonalities and differences in aflatoxin monitoring standards across various countries and regions, giving us a broader international perspective on industry regulations.

Furthermore, the experts shared global practices and pharmaceutical solutions for managing aflatoxin in dairy products. They highlighted the constant emergence of new technologies and methods, driven by advancements in science and evolving market demands, though balancing effectiveness with cost and efficiency remains a significant challenge.

We also presented our biological solution for aflatoxin degradation, which received high praise from the experts. They recognized the innovation and feasibility of our approach and expressed confidence that it could offer a new solution to improve dairy safety. They also provided valuable advice for commercializing the technology, suggesting that we focus on cost competitiveness and the advantages of our method over existing degradation or adsorption techniques. This feedback will be instrumental as we continue to refine our research and plan our market strategy. This exchange not only deepened our understanding of the current state of the dairy industry and aflatoxin treatment technologies but also injected new momentum and confidence into the future development of our project.

--China Chamber of Commerce of I/E of Foodstuffs，Native Produce and Animal By-products (CFNA)

We discussed with experts from China Chamber of Commerce for Import and Export of Foodstuffs, Native Produce and Animal Husbandry (CFNA), Chinese Academy of Agricultural Sciences, Shandong Customs Grain and Oil Testing, Sichuan Kuanwei Food Co., Ltd., and COFCO Shancui Peanut Products (Weihai) Co., Ltd. (in no particular order).

During an in-depth interview with the China Chamber of Commerce for Import and Export of Foodstuffs, Native Produce and Animal By-Products (CFNA), a range of issues concerning the processing, transportation, and safety of food grains were discussed. We inquired about their safety concerns and suggestions for our project.

Experts from the Chinese Academy of Agricultural Sciences posed several questions to us:

1. Whether the suicide system for engineered bacteria is well-designed and if there are any risks to food safety.

2. If the degradation efficiency of aflatoxin by the bacterial strain is sufficiently high.

The interview highlighted the quality assurance measures for grains during processing and transportation. Sealing grains and reducing oxygen levels can effectively maintain the freshness and quality of the grains. Rigorous inspection of grains before processing, removing moldy particles, is an essential step in ensuring food safety. Additionally, maintaining cleanliness and hygiene during transportation is a critical aspect of ensuring food safety.

Control of aflatoxins is an important issue in food safety. Aflatoxins are toxic compounds produced by Aspergillus flavus and pose a serious threat to human health. Countries have established limit standards, such as China's GB2761-2017 standard, which sets very low limits for infant and young child food, demonstrating the importance placed on this issue.

On the technical front, the synthetic biology method for degrading aflatoxin project has received special attention. A student team from Beijing No. 5 High School, participating in the International Genetically Engineered Machine Competition (iGEM), planned to use nanbodies, luciferase, and other means to design a finely controlled device to improve the degradation efficiency of aflatoxins while reducing the impact on the surrounding microenvironment. The advantage of this method is its high specificity and efficiency, precisely targeting aflatoxins for degradation and reducing the environmental pollution and side effects that traditional physical and chemical methods may bring.

Furthermore, the feasibility of synthetic biology methods has been proven in theory. By designing a detection and degradation scheme for aflatoxins through biosynthesis, not only can food safety issues be effectively addressed, but the application can also be extended to various fields such as feed and grains, showing a broad application prospect.

In summary, the design and applicationof synthetic biology methods in the degradation of aflatoxin projects has demonstrated their potential and advantages in the field of food safety. This method can not only effectively solve the problem of aflatoxins in food but also reduce environmental pollution and improve the sustainability of food production. With the continuous advancement of technology and the promotion of applications, synthetic biology methods are expected to play a greater role in the field of food safety in the future.

iGEM is an international competition that attracts many teams from around the world each year. Participating in this competition, the exchange with other teams helps us to better complete our project. To gain the opinions and suggestions of like-minded individuals, we have engaged in extensive communication with other teams, exchanging ideas and suggestions, from which we have greatly benefited.

On August 24, 2024, we participated in a freetalk discussion, where, together with numerous teams from all over Beijing, we each presented our projects and their processes. We took this opportunity to mutually promote the design and improvement of our projects, and to provide the public with synthetic biology knowledge beyond the basic education stage, making the emerging field of synthetic biology more widely known. We also exchanged project ideas and thoughts, and through communication among teams, we enhanced the awareness of unity and friendly cooperation among the various iGEM teams in Beijing.

(A group photo taken during the online project seminar.)

On September 21, we attended an educational seminar organized by the iGEM team from Beijing Institute of Technology. In addition to discussing our projects, we also had an in-depth exchange on synthetic biology and its public outreach. During the seminar, we improved our experimental design to make our projects and results more rational. We also listened to the advice of consultants from the High School Affiliated to Renmin University of China (RDFZ-CHINA), Beijing National Day School (BNDS_China), and Beijing Institute of Technology (iGEM_BIT), absorbing their experience to improve our project. We explored how the paradigms of synthetic biology are reflected in project applications and how high school students can contribute to the development of synthetic biology. Throughout the exchange process, we had in-depth discussions and enthusiastic communication, reaping substantial benefits from one another, and also attracting the interest of other public participants. Among the teams we communicated with, there were many seniors and students who have made significant achievements in the field of synthetic biology. Through our exchanges, we gained not only auxiliary suggestions from various parties but also friendships with many like-minded individuals.

On September 30, the team members of BJWZ-3-China had a multilateral talk with iGEM_BIT and SHSBNU_China to review the obstacles we met and achievements we made in the project. At the same time, we established a new education connection and have further expanded the attraction to the outside world.