Human Practice

(Silver Medal#3, Gold Medal#1)

   Inspired by related news and through offline field trips to farms and hospitals, we learned that there is a problem of antibiotic abuse and the emergence of drug-resistant strains of bacteria, which are closely related to the lives of the general public. In response, we actively explored solutions to the dangers of antibiotic abuse. We have reviewed many literature, interviewed and consulted with many professors and experts in the field to find antibacterial peptides as an alternative to antibiotics, and we have also fully communicated with industry-related departments and companies to improve the production of antibacterial peptides to effectively improve the problem of antibiotic abuse. In the key experimental design steps, we solved the problem of library construction by consulting microbial gene editing experts. During the design and process of the experiment, we also kept close contact with other teams and shared a lot of valuable information with each other. We also conducted an online questionnaire survey and offline public education on synthetic biology in order to investigate the public's awareness and acceptance of antibiotic abuse and antimicrobial peptides. During all the above activities, we always followed the professional's knowledge and ensured the information security of the participants.
   In the whole HP activities, each part is complementary to each other, forming an organic whole.

   Regarding our HP program design, each part of the activity has a clear goal. We make the best use of the HP’s results to improve our project by what we want to achieve. As the project progresses, we divide our HP’s goals into four sections by task type: Background, Public Engagement, Project Design, and Project optimization and improvement, and use these as a guide for our activities. In addition, to ensure that our HP activities were normative and ethical, we consulted with relevant people to guide the design of our activities.


1. The news reflects the social problem of antibiotic abuse

   Our members saw a news article about a duck farmer in Nanjing who abuses antibiotics and never eats ducks himself. The news truly shows the problems in the farming industry today and reflects that the abuse of antibiotics is not uncommon around us, and that antibiotic abuse is not only harmful to human health but also causes serious pollution in the environment.
Fig. 1. Screenshot of news about antibiotic abuse in farms   
Fig. 2. Screenshot of news about antibiotic abuse in the society  
   This news story sparked members' concern about the problem of antibiotic abuse, the seriousness of the problem and the fact that it is literally happening around us, affecting our bodies and our lives. Since then, members of NNU-China have become concerned about the problem of antibiotic abuse and paid attention to good ways to solve the abuse of antibiotics.

2. Listening to the lecture gave rise to the idea of exploring the problem of antibiotic abuse


Fig. 3. Academician Pang Guofang
   Academician Pang Guofang is an expert in the discipline of food science and testing technology, a researcher at the China Academy of Inspection and Quarantine Sciences, an International AOAC Fellow, and a recipient of the International AOAC 2014 Harvey Wiley Award, who has long been dedicated to the research of the theory and practice of food science and testing technology.
   On December 23 2020, Academician Pang came to the School of Food and Pharmaceutical Engineering of Nanjing Normal University to give a relevant lecture on the dangers of antibiotic abuse on human body and the challenges we are facing.
   Through listening to this lecture, we realized the seriousness of the problem of antibiotic abuse and the fact that there are situations around us where antibiotic abuse is causing environmental pollution and harming human beings and nature. So we decided to survey the specific problem of antibiotic abuse and learn about the problem of antibiotic abuse that is closely related to the lives of ordinary people. And we wanted to explore solutions to the problem of antibiotic abuse.
Fig. 4. After the lecture

Public Engagement

3. Research on antibiotic abuse: Visit breeding farm

   On 19 June 2021, members of our NNU-China team visited farmers at Shuangfa pig farm in Baohua Town, Jurong City, Jiangsu Province, to consult on the use of antibiotics in the farm feed and the current feeding and rearing situation, hoping to get a true and objective understanding of the current situation of antibiotic use through field research.
Fig. 5. Feeds in the breeding plant
Fig. 6. Part of the ducks in the breeding plant  
   We were told that , in the last two years, with the increased national attention to the problem of antibiotic residues and the strengthening of policies related to antibiotic use. Therefore, the use of antibiotics in individual users has decreased, but it is still widely used in preventive measures in the relevant sectors. We have also been told that the primary way to prevent pigs from getting sick is daily disinfection, which, while reducing the use of antibiotics, is not quite effective and is still not avoidable.
Fig. 7. A group photo of NNU-China team members with the farm manager

4. Research on Antibiotic Abuse: Visit Jiangsu Provincial People's Hospital

   On 15 July 2021, the NNU-China team visited Dr. Meng Lijuan, Deputy Director of Jiangsu Provincial People's Hospital, to consult with Dr. Meng as well as seeking confirmation that there are many cases of drug resistance caused by the irrational use of antibiotics in the society. We expect to find out whether the phenomenon of drug resistance caused by the irrational use of antibiotics is common through this visit. Moreover, we also wondered if there are good ways to avoid the development of drug resistance.
Fig. 8. Group photo of the team in front of Jiangsu Provincial People's Hospital
   We were told that the current clinical use of antibiotics, just as we thought, adheres to the principles of ‘Only When Necessary’. Antibiotic abuse does exist, but it is not widespread in the hospital now. Generally , antibiotics are only given to patients in acute infections and chronic infections. There are, nowadays, no alternatives to antibiotics in the strictest sense. The foremost thing in the development of antibiotics is to strictly control the use of antibiotics, many of which are used at restricted levels in various countries. Despite the cost of antibiotics is decreasing as a result of national policies, the top antibiotics with high antibacterial power are high-priced as ever.
Fig. 9. Interview with Dr. Meng Lijuan
Fig. 10. Group photo of NNU-China team members with Dr. Meng Lijuan

5. Questionnaire survey on public awareness of antibiotic use

   We distributed an online questionnaire on the issue of antibiotics to the public so as to make certain the current awareness of the public regarding the problem of antibiotic abuse. Also, we looked forward to learn about the level of acceptance and expectation of the public on antibiotic substitutes.
   The number of completed questionnaires was 547. The vast majority of people have a feel for the efficiency of antibiotics, while few people are concerned about the dangers of their abuse, hence we regard that popularizing the detriment of antibiotic abuse is of extreme necessity. Most people, some of them have never even been exposed to them, are in doubt whether they are using antibiotic alternatives.At the same time however, the public are still quite looking forward to having antibiotic alternatives to break the current predicament of antibiotic abuse. Through the questionnaire, we concluded that what the public exactly expects is, under the premise of ensuring quality and safety performance, increasing production to reduce the price of the product.

Fig. 11. Results of the questionnaire survey

6. On-line communication with the Customs officer


Fig. 12. Minister Shen Weijian
   Shen Weijian, male, born in May 1978, senior engineer, member of the Oils and Fats Committee of the Codex Alimentarius Commission of China, member of the Sample Pre-treatment Committee of the Analytical Instruments Branch of the China Instrument Society, member of the Food Professional Committee of Jiangsu Analytical Testing Association.
   On 3 September 2021, on behalf of the NNU-China team, our team leader Xu Yan had a meeting with Minister Shen of Nanjing Customs to seek confirmation on the content of antibiotics in agricultural products, meat and poultry products tested by customs and quality inspection institutes, as well as to learn about the national policies and regulations in this area. We hope to be informed of the current situation of antibiotic abuse through the exchange with Mr. Shen.
   Minister Shen said that at the beginning of his tenure, there is a lot of antibiotics residues in plants and animals, food, but with the improvement of national laws and regulations, each kind of agricultural products and food have clear provisions for antibiotics limits, at present, although can not be completely avoided, the overall antibiotics detection rate has greatly decreased, for food and agricultural products safety showed a great prospect as well.
Fig. 13. On-line communication with Minister Shen

Project Design

1. Interview Professor Jiang Ling


Fig. 14. Professor Jiang Ling
   Prof. Jiang Ling is a PhD supervisor from the Nanjing University of Technology, whose main research interests are the mining and evaluation of new strains of extreme microbial origin and new glycolytic gene resources, rational design modification and catalytic process enhancement of glycolytic enzymes in biocatalytic systems.
   Inspired by related reports and through visits to the farms and hospitals, our team found that antibiotic abuse does exist. In order to effectively solve this problem, we consulted Professor Jiang Ling from the Nanjing University of Technology by email, hoping to learn about methods in the field of synthetic biology to solve the problem of antibiotic abuse through him.
   Professor Jiang Ling pointed out that although there are still other solutions, finding alternatives to antibiotics is still the most effective way to tackle the problem of antibiotic abuse. Prof. Jiang Ling introduced several alternatives to antibiotics, such as bacteriophages and antimicrobial peptides, among which antimicrobial peptides are the alternatives finally identified by our team through literature review. In the end, Prof. Jiang Ling also expressed his great expectation for the success of our project and welcomed us to ask him further questions.
Fig. 15. Email communication with Professor Jiang Ling

2. On-line discussion with GenScript Corporation

Company Profile:

   GenScript corporation provides high-quality biotechnology outsourcing services to researchers engaged in life science research and early stage drug discovery. Its one-stop biological research reagent service platform can provide customers with gene synthesis and related molecular biology services, peptide synthesis services, protein expression and purification services, antibody services and cell line establishment services.
   According to the results of our preliminary questionnaire, the public is looking forward to the availability of inexpensive and safe alternatives to antibiotics. Based on thorough communication with experts and extensive literature review, our team identified "antimicrobial peptides" as an alternative to antibiotics. Therefore, to better understand the problems of antimicrobial peptide production in the current market, we were fortunate to contact the GenScript Corporation in Nanjing and have an online meeting with them on 20 August 2021, in order to study and optimize our expression system as well as to explore the prospects of antimicrobial peptide in biological applications.
Fig. 16. On-line discussions with GenScript
   Through in-depth communication with GenScript Corporation, our team learned that currently there are two sources of antimicrobial peptides: natural extraction and artificial synthesis. From the long-term perspective of time and economy, chemical synthesis is not suitable for commercial large-scale production, but only for scientific research. Microbial synthesis is a promising method, and reducing time and economic cost and improving yield and purity are the key aspects to be considered We proposed our project from a synthetic biology perspective by combining the market and existing technologies: using the mature E. coli expression system to synthesize antimicrobial peptides. Our idea is to construct a library of protein expressing strains with different intensities, transfer the fusion protein of antibacterial peptide and green fluorescent protein into the library, and quickly obtain the best producers through fluorescence screening. Thus, it not only saves time and economic cost, but also improves the output of antimicrobial peptides. GenScript Corporation also approved this idea, and they also expressed their expectation that our project would be successful.

Project optimization and improvement

1. Interview Professor Hao Liu


Fig. 17. Professor Hao Liu
   Dr. Hao Liu, PhD supervisor of Tianjin University of Science and Technology, Tianjin Distinguished Professor. He is the leader of the microbial genetics and metabolism team, and is also the supervisor of Tianjin Microbiology Society and the director of China Biofermentation Industry Association. His research areas are cloning and functional study of important regulatory genes in micro differentiation and metabolic processes, reconfiguration and regulation of microbial metabolic networks.
   Although we've got the information antimicrobial peptides can partially replace antibiotics. The low expression efficiency of antimicrobial peptides is an important problem that puzzles the production of antimicrobial peptides and hinders our research projects. We hope to find a way to increase the expression of antimicrobial peptides. For this reason, we contacted and invited Prof. Hao Liu to give us an interview.
   In the communication with Prof. Hao Liu, we learned that we need to improve the expression efficiency of antimicrobial peptide to solve the problem of low antimicrobial peptide yield. E. coli can be used to systematically optimize the expression. E. coli expression system, a prokaryotic expression production system is recommended to optimize expression rather than simply extracting it from the animal body. Prof. Hao Liu told us the E. coli pET system may be a good choice. But the expression efficency of targetantimicrobial peptides may be low because of the antimicrobial peptides' natural toxicity to bacteria. Furthermore, the expression efficency may vary depending on the different types of antimicrobial peptide. So careful and precise regulation is required to satisfy the production of different antimicrobial peptides.
Fig. 18. Ask Professor Hao Liu for advice on project optimization
Fig. 19. A group photo of NNU-China team members with Professor Hao Liu

2. Interview Professor Xiu-lai Chen


Fig. 20. Professor Xiu-lai Chen
   Prof. Xiu-lai Chen has been engaged in the research of systematic metabolic engineering and synthetic biology for a long time. He mainly undertakes the teaching of the courses of Microbial Metabolic Engineering, Cell Engineering and Organic Acid Processes. He is mainly engaged in the research of metabolic engineering and synthetic biology.
   Because the antimicrobial peptide itself has a toxic effect on the host, the T7 promoter in BL21(DE3) is a strong promoter, it leads to a vicious circle in which excessive expression of AMPs causes a killing effect on BL21(DE3) itself, reducing the number of hosts and decreasing production. We expect to expressed as many AMPs as possible while ensuring host survival by regulating the expression level of T7RNAP.
   To regulate T7RNAP on the translational level, our original design was to construct a Base-Editing tool based on the CRISPR/Cas9 system, and use this tool in editing the RBS of T7RNAP to form the RBS library strains. However, the quality of RBS library constructed by the base editor is not high because of the low diversity. With these problems, we interviewed Professor Xiu-lai Chen and listened to his opinions.
Fig. 21. On-line discussion with Prof. Xiu-lai Chen
   Prof. Xiu-lai Chen said that our project of antimicrobial peptide is well-intentioned, but there are some limitations in using the base editor. The antimicrobial peptide itself is very short, so it is not very convenient if we operate with single gene editing. At the same time, because the vitality of our base editor is not accurately controlled, the effect of applying it to library construction will be poor. Directly use of synthetic degenerate RBS oligos in constructing may be a more efficient option.
   We found that the antimicrobial peptide with the fusion GFP protein was cumbersome, which may hinder the function of antimicrobial peptides. Prof. Xiu-lai Chen pointed out that now the post-processing technology of the fusion proteins can allow us to obtain independent antimicrobial peptides, we need to skillfully design the digestion sites.

Finding the commercial future of the project

Company Profile:

   GenScript is a protein plant with technology development and product development around functional proteins such as enzymes, antigens, antibodies and polymeric organic materials, and has the technology to produce and purify proteins by large-scale fermentation.
Fig. 22. Academician Pang Guofang
   They suggested that the use of antimicrobial peptides is currently used more in farms, especially in aquaculture, but there is no clinical trial in medical treatment, and for the antimicrobial peptides produced by our team with synthetic biology method, in what form can they enter the current market in the future, and their specific commercial plan is still waiting for our further cooperation talk.

   Our HP is not a loose activity independent of the project, but an organic whole that complements each other. All the activities of HP are related to our project design and promotion. With the discussion of team members, we decided the basic idea of HP: to further understand the background to promote the project design through HP, and use the information obtained from HP to optimize and improve the subject design, then guide the experiment through the subject design, and finally evaluate the effect of HP through the test results. At the same time, public opinion is also important to us, so after the project design was perfected to a certain extent, we immediately designed questionnaires to collect social feedback.
   Of course, all our HPs are conducted in strict accordance with the relevant safety guidelines.

I. Discovery background

· How Our HP Inform and Shape Our Project

   To do a good job on a project, the part of preliminary investigation of basic information is crucial. Therefore, after being inspired by the news about the harm caused by antibiotic abuse, we combined the lecture by Academician Pang and went directly to hospitals and farms for field visits to understand the current use of antibiotics. Through our exchanges, our team learned that there is indeed a serious situation of antibiotic abuse and that this has caused some harm to human society, making it particularly urgent to address this issue. This information certainly provided good inspiration for our project that our project is valuable and necessary. And once realized, it will solve social problems and benefit human life.

· How We Respond to Our HP

   Based on information obtained from HP and brainstorming within the team, as well as consulting with professors and experts in the field, we determined the general direction of the project: to find a solution to antibiotic abuse and to implement it effectively.

II. Finding alternatives to antibiotics

· How Our HP Inform and Shape Our Project

   In order to effectively solve the problem of antibiotic abuse and to get concrete solutions, we have met with professors from universities and related companies several times. In each communication, we stick to our own direction and give full consideration to how to improve the positive impact of the project on society and how to benefit more people. Professor Jiang Ling pointed out the search for alternatives to antibiotics as the optimal solution Among them, antimicrobial peptides may be a safe, effective and promising alternative. And through further communication with GenScript, we also learned that the biosynthesis method is more suitable for commercial mass production, with the advantages of reducing time and economic cost as well as increasing yield and purity.

· How We Respond to Our HP

   The information obtained at this stage was the key to the design of our project. We listened to the proposal of Prof. Jiangling and chose antimicrobial peptides as the object of our team's project and conducted in-depth research. We also used the advice given by GenScript to finalize the specific direction of our project: to construct a library of protein expressing strains with different intensities, transfer the fusion protein of antibacterial peptide and green fluorescent protein into the library, and quickly obtain the best producers through fluorescence screening.

III. Project optimization

· How Our HP Inform and Shape Our Project

   In order to optimize and improve the design of the subject content and provide reliable guidance for experimental design, we interviewed expert professors in related fields several more times, and Prof. Hao Liu pointed out that E. coli PET system could be used to systematically optimize expression. Prof. Xiu-lai Chen suggested that we should not be limited to the Base editor system in constructing library, and direct use of synthetic degenerate RBS oligo may be a more efficient choice.

· How We Respond to Our HP

   The HP activities in this session are synchronized with our experiments to achieve the combination of HP and experiments. When problems were found, the HP helped us to solve them. Therefore, the information obtained at this stage was directly reflected in our project design: using of E. coli pET system; direct use of synthetic degenerate RBS oligo in constructing the library.

IV. Results of Our Questionnaire

· How Our HP Inform and Shape Our Project

   The analysis of the questionnaire results provided us with an understanding of social awareness and attitudes toward the dangers of antibiotic misuse and the acceptance of antimicrobial peptides. These analyzed data are consistent with the results of our preliminary interviews with farms and hospitals, proving the relevance and necessity of our study.

· How We Respond to Our HP

   The results of the analysis of these questionnaire data did not directly influence our project design, so we did not have the means to feed it directly as we did with the results of the expert interviews in our design. However, we used this valuable social survey data when establishing partnerships with companies.
   To make the most of the information in the questionnaire, we used it to communicate with companies in biology. Based on the analysis of the information, we were able to reach a consensus that led to a partnership.

Value orientation of the project

   As we move forward and improve the project, we always put safety first. The vision of our project is to use antimicrobial peptides instead of antibiotics to solve the problem of antibiotic abuse in our society. Once these new methods are used on a large scale, any slight problem on safety can become a serious public safety problem. Therefore, the need of safety attention is at the forefront of our program. This was reflected in the questionnaires we distributed to the public, which clearly explained the issue and investigated the public's acceptance.
   When implementing the project, we wanted to protect the interests of our users (hospitals, farms, general public, etc.) to the greatest extent possible. This can be fully reflected in our HP design: we adopted the advice of many industry experts. And during the HP process, we were touched by the real harm caused by antibiotic abuse, which strengthened our determination to solve this problem.
   Because our project uses proven technology and does not directly edit the genes of plants and animals, it does not introduce ethical issues.

Close the loop!

   When running HP campaigns, we focus heavily on translating others' suggestions into the design of our themes, thus creating a closed loop between others' expectations and our designs. These positive feedbacks are shown in the figure below.
Fig. 23. Closed loop between others' expectations and our design