1. Starting point: Anchor the direction from the "dual predicament" of farmers

Xunzi's idea that "knowing is not as good as doing" is the starting point of our project. In the previous experiment on heavy metal remediation by endophytic bacteria (C. metallidurans CML2) in rice, when we entered the rice fields of Lingmi Village, Jiangxia District, Wuhan City, Hubei Province, we heard the helplessness of the farmers: "Sending the soil for testing costs several hundred yuan and takes half a month. When the results come out, the seedlings are already ripe." "Even if arsenic levels are found to be excessive, they dare not use chemical agents for fear of destroying the soil."

Therefore, we have shifted the core objective of the project from "heavy metal remediation" to "arsenic pollution detection + subsequent remediation linkage", and are committed to developing a "low-cost, high-precision, and portable" biosensor.

2. Core Action: A full-chain practice covering "technology - demand - education"

We have carried out five key practices around the entire chain of "ecological hazards - pollution detection - grassroots demands":

3. Methodology: Build a closed loop of "theory - practice - laboratory - society"

Starting from the predicament of farmers, in order to verify the feasibility and necessity of the project and clarify the real needs of the entire human society, our team carried out multi-dimensional research practices covering core scenarios such as expert consultation, experience exchange, questionnaire survey, and science popularization. During this process, we gradually established a "theory-practice system of two-way feedback".

Meanwhile, taking Mendelow's Matrix as a reference, we established a four-quadrant diagram of stakeholders related to arsenic pollution issues. Based on the entire chain of "ecological hazards - pollution detection - grassroots demands", we communicated and connected stakeholders, achieving a collaborative and interactive closed loop of "theory - practice - laboratory - society".

Overview

Our team is always committed to achieving the bioremediation of heavy metals through endophytic bacteria in plants. During a technical practice, we deeply realized that farmers are facing a dual predicament of arsenic pollution: on the one hand, they are unable to detect arsenic pollution in a timely manner; On the other hand, there is a lack of effective repair means. Starting from the demands of farmers, we conducted in-depth research on the current situation and social needs of arsenic pollution. Based on the feedback from the research, we adjusted the technical direction and launched a series of educational and publicity activities. Eventually, we proposed to build a low-cost, high-precision and portable arsenic pollution detection biosensor.

Feedback on Technical Practice challenges

What we did: We went to Lingmi Village, Jiangxia District, Wuhan City, Hubei Province to carry out the "fungus liquid seed soaking" experiment.

Why: We have put the "fungus liquid seed soaking" production method developed by our team into practical application and carried out experiments on the treatment of heavy metal pollution in rice. (This method is based on the endophytic fungus C. metallidurans CML2 discovered by our team in the past. It can alleviate the stress of heavy metals such as cadmium and arsenic and promote plant growth through enrichment.) Moreover, the team has already studied and clarified the cadmium enrichment mechanism of this bacterium and is currently researching its arsenic enrichment mechanism.

What we learned: Farmers in Lingmi Village are facing a double predicament in the production process: 1. It is impossible to obtain the arsenic pollution status of the fields quickly and at a low cost, and there are problems of "expensive and time-consuming" sending to professional inspection institutions. Even if arsenic pollution is detected, there is a lack of "effective and non-destructive soil ecological" remediation methods.

What we adapted to our project: Understanding the difficult demands of farmers in actual production has pointed out the new development direction of our experimental techniques, ranging from the remediation of heavy metals to the detection of arsenic elements.

In-depth exploration of social demands

Professional perspective analysis

What we did: We conducted interviews with key stakeholders in the relevant professional fields: Ke Wenshan (Professor of the College of Life Sciences, Research fields: Plant Ecology, Green Remediation of Heavy Metal Pollution) Cao Yaowu (Lecturer at the College of Resources and Environment, research interests: Water pollution control, groundwater pollution and prevention, contaminated site remediation, migration and transformation of pollutants in underground media).

What we did: We conducted in-depth interviews with Professor Ke Wenshan and Lecturer Cao Yaowu, addressing the practical concerns raised by farmers during the "bacterial liquid seed soaking" experiment in Lingmi Village, such as the high cost of sending samples for testing and the long waiting time for results, as well as the fear that the remediation methods might damage the soil. During this process, we not only provided a detailed introduction to the cadmium enrichment mechanism of the rice endophytic fungus C. metallidurans CML2, but also focused on inquiring about the adaptability of this fungus in arsenic remediation. At the same time, we engaged in professional discussions on issues such as "How to quickly detect arsenic pollution in the field" and "Will the remediation bacteria disrupt the microorganisms in the paddy field?" To provide scientific and reliable practical basis for the implementation and application of plant endophytic bacteria in the biological detection and remediation related technologies of heavy metals (arsenic).

Why: Previously, in Lingmi Village, only the superficial demands of the farmers were understood, but the scientific pain points of arsenic pollution control were not clear - such as the core reasons why the existing detection technologies are difficult to be applied on-site and the key conditions for the survival of remediation bacteria in the fields. All these require a professional perspective to review. We hope that through interviews, we can combine farmers' demands with scientific principles to avoid the subsequent technological research and development being based on assumptions, and to prevent the detection devices developed from being unuser-friendly by farmers or the repair bacteria from becoming ineffective in the fields.

What we learned: Professor Ke Wenshan said that many remediation bacteria work well in the laboratory but fail to adapt to the soil and water conditions in the fields. The key issue is that they do not take into account the "ecological conditions" such as the pH value of the rice field soil and the original microbial community. Our CML2 bacteria need to first test their reproductive capacity in the rice field soil of Lingmi Village; otherwise, the remediation effect cannot be guaranteed. Lecturer Cao Yaowu added that what farmers want about the "arsenic test results" is not just "whether there is or not", but also "whether it is toxic or not" - because inorganic arsenic (As³⁺, As⁵⁺) is more toxic, while organic arsenic is less toxic. The current detection technologies have problems such as relying on large-scale instruments, complex sample pretreatment, high cost, difficulty in on-site detection, and difficulty in distinguishing arsenic forms So being able to distinguish the forms of arsenic is the primary need of farmers.

What we adapted to our project: We combined the information related to arsenic pollution we obtained with the demands of farmers, and incorporated "morphological differentiation" and "ecological compatibility" into the technical goals. On the one hand, when building the arsenic detection research and development plan, we no longer only focused on "low cost", but also needed to find ways to enable sensors to initially identify inorganic arsenic, so that farmers knew "how much dangerous arsenic there was". On the other hand, in the subsequent experiments, we will first designate a small experimental field in Lingmi Village to test the survival and colonization of CML2 bacteria in the local soil, observe the impact on beneficial microorganisms such as nitrogen-fixing bacteria, and ensure that the restoration does not damage the ecological environment of the rice fields. This provides more comprehensive scientific support for the implementation of our technology.

Construct practical theory

What we did:
① Online study the human social practice concepts of outstanding teams from previous iGEM sessions (consult through the iGEM official website);
② Carry out exchange activities with the iGEM team of Wuhan University;
③ Have an interview with Zhang Kai, a senior scholar in the field of humanities and social sciences.

1. Carry out exchange activities with the iGEM team of Wuhan University

2. Interview with Zhang Kai, a senior scholar in the field of humanities and social sciences

Why: In the process of practice, we have realized that we should rely on scientific theories to guide our practice in order to avoid the direction of practice being disorganized. Therefore, we hope to seek theoretical and practical methodologies that can support the advancement of the project through learning, communication and interviews.

What we learned: We understood the core logic of practical theory guidance from different communication partners as follows:
1.From the outstanding teams of iGEM in previous years and the iGEM team of Wuhan University, we have learned that before launching a project, it is necessary to "fully investigate the background", and clearly answer "why to do it" (the necessity of the project), "for whom to do it" (the service targets), and "what value it can bring" (the core benefits). At the same time, it is necessary to achieve "two-way feedback between the laboratory and society" - the experimental plan should be formulated based on social demands, the direction of the experiment should be adjusted according to social feedback during the process, and the experimental results should be fed back to solve social problems, avoiding "working in isolation".
2. From Zhang Kai, a senior scholar in the field of humanities and social sciences, I learned that both theories and things are in a state of dynamic development. When practicing, one must adhere to the core logic of the theory while flexibly applying it in combination with reality. In addition, the feedback during the practical process needs to feed back into the theoretical update, forming a cycle of "theory - practice - feedback - theoretical optimization".

What we adapted to our project: We deeply matched the theories we learned during the communication with the project (the plant endomycete C. metallidurans CML2 for the remediation of heavy metal pollution in rice), and established a "two-way feedback theoretical - practical system" - that is, using theory as a guide to anchor the team's practical direction, ensuring that laboratory research and development are not disconnected from social demands; Through the practice of feedback reverse optimization theory, cognition is dynamically adjusted according to the real scene. A "two-way feedback theory-practice system" has been established, in which theory guides practice and practice feeds back to theory. We also deeply understand that in the process of advancing the project, practice should be taken as the key link. One end connects the technological research and development in the laboratory, transforming theory into practical technical prototypes. The other end connects with the real demands of society, enabling technological innovation to precisely respond to the pain points of human development. Ultimately, a closed loop of collaborative interaction between "theory - practice - laboratory - society" should be formed.

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1. iGEM Experiential Learning

Based on our past team experience and the gains from communicating with the team from Wuhan University, we have compiled the following content around the core of HP (Human Practice) : The opening of the project should be based on thorough background research, and through practical experiences such as "macroscopically anchoring human development needs, meso-level connecting with industry pain points, and microscopically reaching the real feelings of end users", "stakeholder maps", and "analysis of similar iGEM projects", Clearly answer questions such as "What background is the project based on?", "Why is it done?", "For whom is it done?" and "What value can it bring to the development of all mankind?", and drive the team to shift from "determining the technology first and then finding the scenario" to "identifying social pain points first and then matching the technology", optimizing the theoretical framework of research and guidance. In subsequent practice, it is necessary to achieve two-way feedback between the laboratory and society - the experimental plan should be determined and gradually improved based on the social demands investigated in the early stage. During the experiment, real-time communication with society should be maintained, and the direction should be adjusted through social feedback to align with the goal of solving social problems and promoting the healthy and harmonious development of humanity. The experimental results should also be promptly fed back to society to provide new strategies for problem-solving. The entire process avoids "working in isolation", ensuring that the project is always rooted in social needs and serves human development.

2. Humanities and Social Sciences Perspective

During the discussion with Zhang Kai, a senior scholar in the field of humanities and social sciences, the team engaged in a core exchange on "How theory can adapt to the social development and changes in the practical process". In response to the team's concern that "static theories cannot effectively guide dynamic practices", Zhang Kai, taking the evolution of the "social role theory" in sociology as an example, proposed that "the core logic of the theory needs to be adhered to, and specific applications should be flexible." The viewpoints such as "theories and things are in dynamic development" emphasize that in practice, "solving social pain points" should be taken as the anchor point. Through practical feedback, theoretical updates can be fed back to ensure that the project not only conforms to scientific laws but also meets the dynamic needs of human social development.

Understand the needs at the grassroots level

What we did:
① Search for relevant literature and officially released information online (including the World Health Organization, the Ministry of Ecology and Environment and the Ministry of Natural Resources of China, core journals in the field of environmental science, etc.);
② Conduct both online and offline surveys simultaneously (questionnaire surveys are carried out both online and offline, with respondents including college teachers and students, social workers, community residents, etc.)

Why: We hope to focus on the grassroots society, fully and comprehensively understand the actual background and public demands of arsenic pollution, and provide a scientific basis for the exploration of arsenic pollution detection technology. Meanwhile, we hope to carry out a series of educational activities based on the feedback from the research results.

What we learned: We learned about the current situation of arsenic pollution, public demands and technical popular science feedback respectively from literature research, public questionnaires and campus lectures, as follows:

1. The severity of arsenic pollution worldwide and in China (based on official reports and literature reviews) :

At the global level: Arsenic has been listed by the World Health Organization (WHO) as the top of the "10 Chemicals of Major Public Health Concern". Its main threat comes from contaminated groundwater - inorganic arsenic is a Group 1 human carcinogen. Long-term exposure can induce skin cancer, lung cancer, bladder cancer, and also hinder children's cognitive development and increase the risk of cardiovascular diseases. According to data from the WHO, at least 70 countries around the world have about 140 million people drinking water with arsenic content exceeding the safety standard of 10μg/ L. In regions such as Bangladesh and West Bengal in India, the arsenic content even exceeds 500μg/L, creating a severely affected area of "arsenic poisoning from drinking water" [1].

At the Chinese level: According to the "Bulletin of the National Soil Pollution Status Survey" (Ministry of Ecology and Environment, Ministry of Natural Resources, 2014), the rate of arsenic content exceeding the standard in soil in China is 2.7%, and the rate of arsenic content exceeding the standard in cultivated land is 19.4%, making it one of the main inorganic pollutants in cultivated land [2]. Pollution shows the characteristics of "heavier in the south and lighter in the north, with concentrated mining areas" The antimony and arsenic mining areas in the southwest (Yunnan and Guizhou), the heavy metal smelting areas in the central south (Hunan and Hubei), and the industrial-intensive areas in the Yangtze River Delta are high-incidence areas. Among them, the arsenic content in the soil of the antimony mining area in the southwest exceeds the "Soil Pollution Risk Control Standards for Construction Land" (GB 36600-2018) by 3 to 10 times, reaching "extremely strong ecological risk" [3]. Meta-analysis of farmland soil in the past 10 years shows that the accumulation rate of arsenic in rice fields in southern China is significantly higher than that in northern China. In some production areas, the arsenic content in rice exceeds the limit of 0.15mg/kg stipulated in the "National Food Safety Standard - Limits of Contaminants in Foods" (GB 2762-2022), directly threatening food security [4].

2. Core public demands for arsenic pollution and detection technologies (based on online and offline questionnaires) :

Cognitive level: Over 60% of the respondents have insufficient understanding of the sources of arsenic pollution (such as mineral mining and arsenic-containing pesticide residues) and exposure routes (such as consuming arsenic-contaminated rice and drinking contaminated groundwater), and only 23% can accurately identify the early symptoms of arsenic poisoning (such as skin pigmentation and keratinization).

At the level of testing requirements: 85% of the respondents are concerned about the rapid detection technology for arsenic elements. Their core demands are mainly focused on "low cost" (avoiding the single detection fee of several hundred yuan for professional institutions), "high portability" (supporting operation in field and home scenarios without the need for large instruments), and "fast speed" (hoping to get results within 1-2 hours). This is completely in line with the previous pain point of the farmers in Lingmi Village, who were unable to quickly and at low cost learn about the arsenic pollution situation in their fields. -

At the technical expectation level: Over 70% of the respondents have little knowledge of "biosensors", but 92% expect their application in fields such as medical and health care (such as arsenic exposure monitoring in the body) and food testing (such as arsenic content screening in rice), providing a direction for the project to explore a "repair + detection" linkage solution.

What we adapted to our project:

1.Based on the feedback from the research and presentation results, and through the joint brainstorming of the team members, we have decided to respond to the grassroots demands and develop a low-cost, high-precision, and portable arsenic pollution detection biosensor.
2.In response to the public's insufficient awareness of arsenic pollution, we decided to conduct popular science education on the background of arsenic pollution through campus lectures and science popularization. At the same time, we introduced the huge potential and value of biosensors, further stimulating their enthusiasm. It has laid a potential cognitive foundation for the subsequent technical promotion of the project.

The solution has been established and perfected

The determination of the project solution is not a "single decision". We comprehensively considered the opinions of experts in relevant fields, technical feasibility and social demands, and ultimately decided to develop a single-cell biosensor that can convert arsenic pollution into visual fluorescence signals, thereby meeting the grassroots demands of "low cost, portability and real-time detection" in arsenic pollution detection.

1. Interview with Professor Zhang Haimou

Member of the Teaching Steering Committee for University Biology Courses of the Ministry of Education, Member of the Education Professional Committee of the National Biochemistry and Molecular Biology Society, Standing Director of the Hubei Synthetic Biology Society, Standing Director of the Hubei Toxicology Society, Standing Committee Member of the Wuhan Municipal Committee of the Chinese People's Political Consultative Conference, Vice Chairperson of the Wuhan Municipal Committee of the China Democratic League. Main research fields: Biology education, environmental toxicology

What we did:
We interviewed Professor Zhang Haimou with two core questions in mind: First, what kind of chassis cells should be selected in the initial stage of the project - should we continue to use our rice endophytic bacteria CML2 or choose the commonly used engineering bacteria in the laboratory? The second question is whether the sensor system should be "single-cell" or "dual-cell"? During the interview, we elaborated on the cadmium enrichment effect of CML2 and our goal of developing an integrated "detection-repair" bacterial agent. We also expressed concerns such as "CML2 has not been modified much, so will the sensors be unstable?" and "Will the two-cell system be too complex for farmers to use?"

Why:
When we were testing the sensor in the laboratory before, we found that the expression of the exogenous gene of CML2 fluctuated up and down, and there were always problems with the signal transmission of the two-cell system. We were afraid that we would take detours if we continued to do so. We aim to determine a reliable technical route through the professor's professional judgment - for instance, which chassis cells can reduce research and development risks and which system is more suitable for later field application, to avoid the situation of "working hard for a long time but not being able to use it in the end".

What we learned:
Professor Zhang said that CML2 has not undergone systematic engineering transformation at present. It does not meet the "core indicators of the chassis" such as cell activity stability and the efficiency of exogenous gene expression. If it is directly used in the initial stage, it is very likely to cause large fluctuations in sensor data, even construction failure, and increase research and development costs. It is suggested that we give priority to using Escherichia coli DH5α - this bacterium has a clear genetic background, simple culture conditions, and is stable after the introduction of exogenous genes. It is suitable to first build a "test platform" to quickly verify whether the sensor solution can succeed. Once the data is sufficient, we can then decide whether to modify CML2 or use it for subsequent development. Regarding system selection, he mentioned that the two-cell system can theoretically complement each other, but in practical operation, it involves co-culture of cells and cross-cell signal transmission, which is a very complicated process. If we have no experience, we are very likely to get stuck. It would be better to focus on developing the single-cell system first, to run the complete functions of "arsenic recognition - signal conversion - fluorescence emission" smoothly and ensure stability, and then consider adding the dual-cell function.

What we adapted to our project:
We immediately adjusted our technical route First, use Escherichia coli DH5α as the chassis to build a single-cell arsenic detection sensor, focusing on verifying whether "fluorescence can be stably produced after arsenic addition" and "whether the fluorescence intensity can correspond to the arsenic concentration". Once this basic function is successfully run and the data is stable, then evaluate the modification direction of CML2 - such as how to make the expression of its exogenous genes more stable Can it be compatible with the loop of single-cell sensors? At the same time, the "dual-cell system" is temporarily listed as a "long-term goal". At this stage, we will not be distracted. We will first solidify the single-cell technology that can be implemented to avoid delaying the project progress due to high complexity and ensure that the technology for farmers in the later stage is stable enough.

2.Interview with Professor Dai Longhai

Research fields: Biocatalysis and enzyme engineering, enzyme structure and catalytic mechanism

3.Interview with Professor Vanessa

(Hubei Microbiology Council Member, Hubei Synthetic Biology Society Council Member, Hubei Biochemistry Society Council Member, Young Editorial Board Member of BioDesign Research. Research fields: Molecular enzymology, biosensing, etc.)

What we did:
We interviewed Professor Vanessa with the question of "insufficient sensitivity" of the sensor - for instance, when low concentrations of arsenic (0.05mg/kg) are added, the fluorescence is very weak and almost invisible to the naked eye, and farmers might mistakenly think it is "safe". In the interview, we elaborated on the single-cell circuit design of the sensor: using the ArsR repressor protein to recognize arsenic and then activating GFP to express fluorescence. We also mentioned that we had tried adjusting the amount of the repressor protein, but the effect was not significant. We would like to ask how to improve the sensitivity and whether the natural promoter needs to be modified.

Why:
Insufficient sensitivity is a major issue - some fields in Lingmi Village are slightly contaminated with arsenic. If the sensor fails to detect it, farmers will not use restorant agents, and in the end, the rice may still exceed the standard. We want to know where the problem lies. Is it due to insufficient repressor proteins, or is the promoter not strong enough? Or are there other optimization directions to ensure that the sensor can detect mild pollution and help farmers detect and deal with it early?

What we learned:
Professor He said that our problem is very likely to lie in the expression level of the repressor protein - the repressor protein is a "key component" for recognizing arsenic. If the amount is too small, low concentrations of arsenic cannot be promptly "triggered" for fluorescence expression, and the natural fluorescence will be weak. She suggested that we first screen the constitutive promoters upstream of the repressor protein: promoters of different intensifiers will result in different expression levels of the repressor protein. For instance, strong promoters can produce more repressor proteins, and low concentrations of arsenic can also quickly bind and initiate fluorescence, thereby enhancing the sensitivity. She also mentioned that natural promoters and the repressor proteins and GFP we use might not be compatible - for instance, if the promoter is too strong and there are too many repressor proteins, it may instead suppress fluorescence. It is too weak and insufficient. In the later stage, it may be necessary to modify the promoter through site-directed mutagenesis and other methods to make it better compatible with the protein and ensure stable sensitivity.

What we adapted to our project:
We immediately arranged the "Constitutive Promoter Screening Experiment" : Three different intensifiers of promoters (weak, medium and strong) were identified and respectively connected to the front of the repressor protein gene to create three sensor prototypes. Then, the fluorescence intensifiers at the same low concentration of arsenic (0.05mg/kg) were measured. The results showed that the prototype of the medium-intensity promoter had the most obvious fluorescence, which could be clearly seen with the naked eye, and the sensitivity just met the standard. Subsequently, we will define this medium-intensity promoter as the "basic model", and simultaneously record the fluorescence data of different batches of sensors. If any sensitivity fluctuations are detected, we will, as Professor He mentioned, attempt to modify the key sites of the promoter to further optimize the compatibility and ensure that the sensor can stably detect low concentrations of arsenic.

4. Interview with Professor Hu Yumei

Research directions: Biocatalysis and enzyme engineering, enzyme molecule modification, analysis of enzyme structure and catalytic mechanism