Team:SCUT-China/Integrated


1. Problem formulation

1.1 Bites

South China University of Technology is located in the humid and rainy Guangzhou, a warm climate with four indistinguishable seasons not only suitable for human habitation, but also for mosquito breeding. Whether indoors or outdoors, the mosquito problem has always plagued us, and other people living in Guangzhou. According to the data of Baidu index, from 2013 to 2021, the users who search the keyword of mosquito repellent on the Internet are most from Guangdong. And among Guangdong, the most dominant search users are from Guangzhou. The searches include recommendations of mosquito repellent methods and evaluations of various means of repelling mosquitoes, which can show that the problem of mosquito repellent is a high concern and urgent problem for people in Guangzhou, as well as the known methods of repelling mosquitoes that people can't meet their needs.

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    Fig.1-2 provincial and city ranking of Search times
    http://index.baidu.com

    1.2 Mosquito-borne disease:

    Mosquito bites bring not only itchy skin, but also the risk of contracting mosquito-borne infectious diseases. Mosquitoes are a vector organism and diseases that use mosquitoes as vectors include yellow fever, dengue fever, epidemic B encephalitis, malaria, lymphatic filariasis, Chikungunya, West Nile virus disease and Rift Valley fever.

    Fig.3 Mosquito-borne diseases
    Source:https://www.ucdavis.edu/news/

    The most direct solution to vector-borne mosquitoes is the use of insecticides. However, surveillance has found that insecticide resistance is widespread among malaria vectors. According to the World Health Organization, 65 of 89 malaria-endemic countries have reported at least one endemic vector resistant to pyrethroids since 2010. All four classes of insecticides currently used for adult malaria vector control, pyrethroids, organochlorines, carbamates, and organophosphates, have become worldwide. In addition, malaria vectors in some parts of Africa are now able to survive in environments with high concentrations of insecticides, suggesting that resistance in some malaria vector organisms is now increasing over time.

    Fig.4 Insecticide resistance all over the world
    Source: WHO Malaria Threats Map.

    2.The definition of Human Practices

    Before starting the task, we first had to understand the main purpose and nature of the task. Therefore, we first conducted a search of human practice to enhance our understanding of it through cutting-edge literature research. According to Liu's research [1], the contradiction between the real world and human’s needs constitutes the problem to be solved by human practice, and transforming reality is the central task of human practice. That is, the main purpose of human practice is to change the conflict between the actual situation and human’s needs.

    However, the actual situation and human’s needs are constantly moving forward and changing [2], so human practice is also a process of change. Firstly, the existing conflicts between the actual situation and human needs are solved through practical activities, then the changes in the actual situation and human’s needs are recognized after solving the existing conflicts, and then the new conflicts arising from them are solved through practical activities, and so on and so forth. Therefore, human practice must be an infinite development process of dialectical movement from practice to knowledge and then from knowledge to practice in a spiral and wave-like advance.

    Therefore, we simplified the main purpose and essence of human practice based on the knowledge about human practice by the following representation method with logic gates and genetic lines. Where the solid line indicates that there is proof in the literature and the dashed line indicates our vision of the future task.

    Fig.5 The definition of Human Practice

    After defining the problem we want to solve, we need to both know what people need and follow the basic principles of practical situations in our practice.

    To know people's needs, it is first necessary to determine who these people are, that is, to identify the stakeholders of our project. Then it is about understanding the methods of knowing their needs such as communication through interviews, questionnaires and focus groups and the techniques of how to better know their needs such as using different interview techniques for different groups.

    Practice is about solving existing conflicts, and in the case of our project, making our project better and more likely to have real-world applications. Talking to relevant cutting-edge researchers allows for suggestions to make the basis and core of the project - the experimental design - more rational. And through computer modeling, complex problems are made simpler, providing help to reduce the workload of experiments.

    In practice, it is important to recognize real-world realities such as the current state of mosquito repellent methods and product use. However, we also need to follow some basic principles, such as considering the safety of the project, ethics and our own sense of conviction to make the world a better place.

    In addition to making projects that ultimately resolve the conflicts of stakeholders better, it is education that allows them to understand the realities of the conflict and how to resolve it during the communication process, increasing the likelihood that in the future they will also have the ability to resolve it.

    2.1 Stakeholder identification

    Stakeholder theorists argue that stakeholder relationships should be based on the principles of fairness and reciprocity [3]. It has been noted that the traditional research paradigm of treating research subjects or targets of project development as passive audiences may lead to research findings that are not well aligned with the information needs of real-world decision makers [4,5] . Therefore, we should first identify stakeholders, then assess their skills, knowledge, and expertise, and determine their needs, interests, and expectations as a way to determine the level of stakeholder involvement in the overall project. Moreover, the part of identifying, engaging stakeholders must start far in advance so that stakeholder input is considered an important part of the project implementation [6] .

    To better identify stakeholders, we need to know its definition. Freeman defines stakeholders as, “any group or individual who can affect or is affected by the achievement of the organization's objectives”. According to the Project Management Institute (PMI), the term“stakeholder”refers to “an individual, group, or organization, who may affect, be affected by, or perceive itself to be affected by a decision, activity, or outcome of a project” [7] . That is, almost any individual or group that has or perceives to have a stake in a consensus-building process will be affected in some way, positively or negatively, by the execution of the project as a result of the work or its outcome, and ultimately may also affect the outcome of the project or the ability of that project to have a positive or negative impact.

    However, the definition of a stakeholder as articulated in different stakeholder theories is currently controversial. As an inherently controversial concept, the solution lies not in a universal definition of stakeholder but in a debate on the boundaries of stakeholder identification. Samantha Miles [8], through keyword extraction, definition analysis, classification, filtering and ranking of 593 different stakeholder theories defining stakeholder theories and stakeholder definitions, produced a comprehensive, multidimensional classification of stakeholder theories. The structure of the classification model also juxtaposes the existing stakeholder theories and their applications in multiple contexts.

    Fig.6 The definition of different stakeholders

    According to the essay, an influencer stakeholder is an individual, or group that has the capacity to influence the actions of an organization and has an active strategy to do so. A claimant stakeholder is an individual, or group that has a claim on an organization and an associated active strategy to pursue the claim but lacks the power to guarantee that the claim is attended to by management. A collaborator stakeholder is an individual, or group that co-operates with an organization but lacks an active interest to influence the organization. A recipient stakeholder is an individual or group that is a passive recipient of the impact of organizational activity.

    That is, joint consideration of the effects of power, interest, and influence allows for the classification of stakeholders into four categories, including influencers who can actively influence the strategic development of a project, collaborators who passively receive influence, claimants with potential influence, and recipients who do not influence the project.

    Since we are primarily addressing mosquito problems and their associated insecticide resistance and mosquito-borne infections, our project stakeholders include: governments and organizations currently responsible for addressing these problems, professionals working on related issues or projects, factories producing related products or processes, and end-users of related products.

    Influencer stakeholders should be managed closely and continuously updated. Collaborator stakeholders should be kept informed about the project. Recipient stakeholders should be monitored, and claimant stakeholders should be kept satisfied.

    According to the theory of Samantha Miles [9], the following classification of our stakeholders can be generated.

    Fig.7 The classification of our stakeholders

    2.2 Interviewing Techniques

    Interviewing is currently the main approach taken by iGEM teams to conduct human practices, which should take different measures and interviewing techniques for people with different identities and personalities. Considering the relatively large cultural differences between countries and states, our stakeholders are also mainly Chinese. Therefore, we most refer to the relevant literature published by Chinese scholars and the experiences shared by Chinese TV journalists.

    In order to better inspire other Chinese iGEM teams, we conducted a summary report on the standardization of the interview process and the interviewing techniques for different populations. While writing this report, we had difficulties in defining the different identities, so we consulted with Professor Ning Qunxian, who has extensive interviewing experience in the School of Journalism and Communication at South China University of Technology.

    In addition to this, we also determined the standardization of the interview process we set up, consulted on the selection of interview timing, techniques for interviewing different people and writing the manuscript, and ways to secure information. Due to the restrictions of the epidemic, many teams can only conduct interviews online at the moment, so we also made some suggestions on how to conduct online interviews.

    Click here to know more about the interview

    Click here to know more about Interviewing Techniques

    3.How others solve the problem

    Whether it is the problem of common mosquito bites, the spread of mosquito-borne infections or insecticide resistance is an urgent problem that needs to be solved and has been studied for many years. Therefore, before we start to conceive our solution, we should first understand the existing mosquito control methods, and understand how the current stakeholders are considering to solve this problem.

    3.1 WHO

    “Vector behavior that compromises the effectiveness of core interventions must be tackled through the use of new tools.”

    Mosquito-borne diseases, which are naturally epidemic in origin, are transmitted by mosquitoes. Since the prevention and control of mosquito-borne diseases have certain commonalities, we mainly refer to the technical prevention and control guidelines for malaria, one of the world's deadliest mosquito-borne diseases, written by World Health Organization.The Global Malaria Technical Strategy 2016-2030 aims to help countries reduce the human suffering caused by this world's deadliest mosquito-borne disease. Different countries should take different measures depending on their national situation. The strategy was adopted by the World Health Assembly in May 2015. The strategy are divided into three pillars according to the level of malaria elimination, from universal prevention, diagnosis and elimination of malaria in pillar 1, to measures recommended to achieve malaria-free status in pillar 2, to measures recommended to avoid re-emergence of malaria due to imported cases in pillar 3. With surveillance at the core, WHO provides comprehensive technical guidance to countries and development partners for the next 15 years, emphasizing the importance of scaling up the malaria response and eliminating malaria.

    According to the WHO, after seven decades of effort, China has become a malaria-free country. That is to say, China is now in pillar 3 in terms of the development of malaria as a disease - we should shift from malaria surveillance to core interventions.

    Fig.8 The summary of the report

    Fig.9 The screenshot of the news

    In the paper, we also found that for our concerned issue, insecticide resistance, the main approach currently taken is to use insecticides with different modes of action, through cyclical changes between indoor residual spraying rounds (rotation) or multiple combined interventions. Still, vector behavior that affects the effectiveness of core interventions must be addressed through the use of new tools, and the cost of vector control products is a major obstacle to the implementation of strategies to prevent and mitigate resistance and reduce residue transmission, which reinforces our confidence in the development of new, cost-effective tools.

    3.2 Guangdong Center for Disease Control and Prevention (1/2)

    “The creation of a new drug may require thousands of dollars of investment. But it may only take one or two years for a drug resistance to develop.”

    The strategy is very important, but the practical process often encounters more problems. Thus after learning about WHO's strategy for the future of malaria, we decided to consult with the in Guangdong Province, the province where Guangzhou City is located, to understand the current situation of mosquito-borne diseases in Guangdong, the government's investment in mosquito-borne disease surveillance, and the main measures taken in China.

    In the process of introducing the basic situation of mosquito-borne diseases in Guangdong Province, we learned that the dengue fever, which should be mainly produced in the south of China, broke out in Shandong in 17 years, probably due to the warming climate and the northward shift of the distribution of Aedes aegypti. This made us realize that mosquito-related science not only needs to be carried out in the south, but also in the north, so we cooperated with Jilin University, which is also preparing to go into the field of mosquito repellent, to promote science promotion, so that more people in the north can see our WeChat passages.

    In terms of the current focus of mosquito-borne disease control, the main economic investment of the provincial CDC is in the monitoring of mosquito-borne infectious diseases, including reagents and sophisticated instruments. In addition, the province's sentinel hospitals will report the number of tests and positives in real time, as well as hospitals to provide regular training to health care workers on how to diagnose symptoms of mosquito-borne infectious diseases. As far as the basic prevention method is concerned, it is based on the removal of mosquito breeding sites such as stagnant water, supplemented by chemical agents for the elimination of adult or larval mosquitoes. Combined with the information we have collected, it shows that for mosquito-borne infectious diseases, Guangdong is currently implementing mainly case, vector surveillance and vector control combined with patriotic health campaigns as the main prevention and control measures.

    In addition, experts also stressed the seriousness of the current domestic insecticide resistance, as well as the possibility that new pesticide research and development investment and effectiveness is not proportional due to the rapid development of drug resistance.

    Through this interview, in addition to the basic situation of mosquito-borne diseases and prevention and control measures in Guangdong Province, we also learned about the importance of surveillance for the prevention and control of mosquito-borne infectious diseases and the difficulty of solving insecticide resistance.

    In order to understand more about the actual situation related to mosquito-borne disease detection, we collaborated with Jilin China and together we interviewed Kan Bo, a laboratory doctor at the Second Hospital of Jilin University. We did not travel to Jilin to conduct the interview in order to cooperate with the epidemic control, but communicated with the Jilin University team member in charge of the interview in advance about the purpose of the interview and the main content of the interview.

    Click here to know more about the interview

    3.3 Laboratory physician

    While browsing the WHO webpage on malaria, we discovered a gene, the Plasmodium falciparum histidine-rich protein 2 gene (Pfhrp2), which is one of the important tools for Plasmodium detection, the deletion of which can lead to false negatives in which Plasmodium falciparum is difficult to be detected. To investigate what to do to break this dilemma, we started the next step of our investigation.

    Dr. Kan Bo, a laboratory physician from the Second Hospital of Jilin University, first affirmed the importance of insect-borne infections in preventive medicine, and then he introduced us to the current preventive measures for mosquito-borne diseases, including strengthening the monitoring of insect and epidemic sources to and from and close contacts, carrying out sanitary environment management work and mosquito elimination. Dr. Kan also emphasized the importance of new drug development to reduce insecticide resistance.

    There are different testing methods for different mosquito-borne diseases, and they are all relatively mature now. For the solution of the problem of false negative Plasmodium test, Dr. Kan proposed multiple retests and genetic diagnosis. In addition, he also mentioned the personal protective measures that the general public can take against mosquito-borne diseases, including spraying mosquito repellent to prevent mosquito bites when going out and cleaning up areas with stagnant water in the living environment.

    This provides us with another way of thinking. While monitoring and detection are important, these are long-term strategies. We believe that addressing mosquito bites is a more effective measure of personal protection for the general population and can be put into use relatively quickly. Consequently, we have shifted our focus from monitoring and detection to mosquito repellent products.

    4.How we expect to solve this problem

    4.1 How can we design a project that can be put into widespread use as soon as possible to benefit the general public who are suffering from issues relevant to mosquito and be able to performed by individuals effectively?

    4.1 Questionnaire

    We released a questionnaire to get the types of mosquito repellent products people commonly use and the characteristics they think mosquito repellent products need to have. The number of people who completed the questionnaire was 224, including 60 parents of young children or the pregnant. More than 80 percent of people would choose mosquito repellent spray for children and themselves outdoors, and most of them would consider safety as the most important factor when choosing mosquito repellent products. In addition to safety, they are also concerned about effectiveness, pleasant odor, skin sensitization, brand, portability and price.

    Fig.10 Choice of mosquito repellent methods outdoors

    Fig.11 Choice of mosquito repellent methods outdoors

    4.2 Forestry workers

    People who often need to work outdoors especially in places with a lot of plants are also the people we need to consider. Therefore, we worked with Jilin China to interview some technicians and front-line workers from Xingan County Ganjiang Chinese Herbal Medicine Co., Ltd. in Jiangxi Province.

    We first asked him about his daily work, which is all outdoor work such as weeding, fertilizing, and agricultural planting. Although he thinks their group is seriously troubled by mosquito bites, especially when working in a lot of plants and cold and wet environment, he seldom uses mosquito repellent spray because all of them he has used basically have short effective time, poor repellent effect, and smell bad when combined with sweat. Thus, he hopes that in addition to improving the effectiveness and duration of mosquito repellent, it can also have a certain fragrance. We also consulted his main access to knowledge related to mosquito-borne diseases, mainly short video software such as Tiktok. This provides some suggestions for the selection of media for our science-related knowledge.

    Fig.12 Jilin-China is interviewing forestry workers

    Combining the above surveys and interviews, we chose mosquito repellent spray as the research vehicle to find mosquito repellent ingredients that are simultaneously safe, effective, long-lasting, fragrant and non-skin sensitive.

    4.3 find out Nootkatone

    DEET, IR 3535, Picaridin and Oil of Lemon Eucalyptus are currently considered recommended mosquito repellents. But these ingredients have certain disadvantages. For example, DEET can cause skin rashes at higher concentrations [9]. Besides, it also leads to reduced sun protection when used with sunscreen [10]. IR 3535 and Oil of Lemon Eucalyptus are lacking in safety, as they can cause greater eye irritation [11,12], and IR 3535 may cause corneal opacity. In contrast, direct use of oil is not recommended for people with sensitive skin. And Percocet is not very popular among consumers in the market due to its high price.

    In the EPA's report on new repellent ingredients, we note the high and stable long-term repellent effect of nootkatone compared to existing repellent ingredients. The data from the EPA proves that not only is the high-purity nootkatone highly safe, it is also non-irritating, non-skin sensitizing and a qualified mosquito repellent ingredient.

    4.4 Confirm the safety of Nootkatone

    We therefore summarize the species of bugs that can be repelled/killed by nootkatone. It can be seen that nootkatone is effective for many species and has potential as both a repellent and an insecticide. References are marked at the back of the species, so you can check there for more specific information.

    On the question of whether or not Nootkatone can provide some relief from insecticide resistance, we asked IRAC and received a response. First of all, new chemical insecticides are still necessary in the presence of a technology like transgenic mosquitoes. Due to ethical issues, it is controversial whether genetically modified mosquitoes can be put into use, and while this technology can prevent the spread of mosquito-borne diseases, it cannot completely replace insecticides. Then, the current slow progress in the development of mosquito repellent products is mainly because the market is small and the return on investment is small, so the possibility of drug resistance is relatively low. And they mentioned a very important point is that it is difficult to find both safe and effective repellents, and repellents are almost no resistance to produce. However, they are unstable and need to be sprayed regularly, thus posing a challenge to the control of mosquito-borne diseases on a large scale. Finally, they give us a hint that we can determine the repellent effect of Nootkatone by dose response and then repeat the experiment with mosquitoes that are already resistant to a particular insecticide, and if the results are not very different, then cross-resistance is unlikely to develop between the two.

    Click here to know more about Nootkatone

    Fig.13 The summary of the effect of Nootkatone

    4.5 IRAC

    IRAC is an international insecticide resistance organization, and to find out whether or not Nootkatone really has the potential to address insecticide resistance, we consulted with Mark Hoppé, Chair IRAC Public Health team by email.

    On the question of whether or not Nootkatone can provide some relief from insecticide resistance, we asked IRAC and received a response. First of all, new chemical insecticides are still necessary in the presence of a technology like transgenic mosquitoes. Due to ethical issues, it is controversial whether genetically modified mosquitoes can be put into use, and while this technology can prevent the spread of mosquito-borne diseases, it cannot completely replace insecticides. Then, the current slow progress in the development of mosquito repellent products is mainly because the market is small and the return on investment is small, so the possibility of drug resistance is relatively low. And they mentioned a very important point is that it is difficult to find both safe and effective repellents, and repellents are almost no resistance to produce. However, they are unstable and need to be sprayed regularly, thus posing a challenge to the control of mosquito-borne diseases on a large scale. Finally, they give us a hint that we can determine the repellent effect of Nootkatone by dose response and then repeat the experiment with mosquitoes that are already resistant to a particular insecticide, and if the results are not very different, then cross-resistance is unlikely to develop between the two.

    5. Project design: how to achieve high yield of Nootkatone

    5.1.Determine the chassis

    After determining to increase Nootkatone by synthetic biology, we started to think about which chassis cells to choose. By reviewing reviews and research articles on biosynthetic terpenoids, we compared the advantages and disadvantages of several "factories" in detail, and finally determined our factory -- Saccharomyces cerevisiae.

    5.2.Background introduction

    Studies have shown that S. cerevisiae has the Mevalonate pathway (MVA). Valencene synthase (VS) catalyzes the synthesis of Valencene from MVA metabolite FPP. Then Valencene can be oxidized by HPO, CPR and ADH to synthesize Nootkatone. Our PI is Professor Shuang Li from South China University of Technology. Her research group has at least five years' research foundation in the biosynthesis of Nootkatone. The previous research of our group has successfully improved its synthesis in S. cerevisiae cells by enhancing MVA pathway and knocking out or weakening FPP pathway. However, the current maximum yield has room for further improvement and is far from meeting the demand for Nootkatone, a high value-added product.

    In order to come up with strategies to further increase production, we contacted Suzhou BIOSYNTHETICA, an emerging enterprise in biological manufacturing. In the communication with the experts, we learned some important information. For the cell factory we constructed to produce Nootkatone, the allogenic metabolic pathways we added would compete with the cell's own metabolism for limited energy and substances in the cell growth stage, while the later stage is an important period for the production of target products. If we use the inducible promoter, we can induce the glucose metabolic flow to acetyl-CoA as much as possible, and let acetyl-CoA be used for the growth of cell in the early stage, and the production of target product in the later stage. Therefore, the expression level of related genes at the later stage of fermentation plays a key role in product accumulation. If the gene does not maintain a high expression level at the later stage, the golden moment of mass accumulation of target products may be missed.

    Excitingly, this has also been confirmed in our literature research. In our in-depth understanding of chassis cells, we learned that the fermentation of S. cerevisiae has the "Diauxic shift", and most of the natural promoters of S. cerevisiae are associated with Glycolytic genes. The difference in the expression level between the pre-fermentation and post-fermentation stages will greatly reduce the efficiency of our cell factories.

    Then, we discussed these exciting gains with our PI. PI mentioned that when the strong promoter TEF1 commonly used in yeast was used to express Valencene synthase, the yield of Valencene was not high, which was probably related to the Diauxic Shift and Crabtree effect we mentioned. In addition, PI also told us that promoter libraries with a wide dynamic range are of great help for fine regulation in metabolic regulation. Therefore, we focused on the promoter modification of S. cerevisiae.

    We started the painstaking literature research. Although it was hard, it was also fruitful. We were inspired by a 2019 paper in ACS Synthetic Biology in which Arun S. Rajkumar et al, based on the "Diauxic shift" of S. cerevisiae, demonstrated a reversal of regulatory function in two glycolytic yeast promoters by replacing glycolytic regulatory elements with ones induced by the diauxic shift to satisfy the need of alternate carbon sources or a need to decouple growth from production. We then applied similar ideas to our wet lab design. According to our production requirements for cell factories, we need to find a trigger to activate gene expression at the ethanol utilization stage, so as to maintain high intensity. Like Arun S. Rajkumar et al, diauxic Shift can be used as a good trigger to make promoters with inductivity without the need for additional inducers to interfere with cell growth.

    In the promoter search phase, our goal is to find a batch of yeast promoters with potential and screen promoters for later transformation. At the beginning, our idea was to screen promoters in other types of yeast to obtain greater possibilities. However, through communication with experts from Suzhou BIOSYNTHETICA, we learned that the replacement of regulatory elements across subspecies was highly unstable, and it was not recommended to consider such a scheme in the early stage of the project. So we limited our search to S. cerevisiae finally.

    In wet lab, molecular cloning experiment was a big problem for us due to our lack of experience. At first, we used enzyme digestion and ligating method to construct promoter-VS-SAG1T expression cassette, but PSOL4 could not connect to the vector due to sequence specificity. After consulting with our advisor, we switched to homologous recombination, which is a seamless new method that can avoid some of the sequence specificity problems to a certain extent and quickly solved this problem we met.

    With the process of the project, we inevitably encounter the problem of industrialization. To make sure our project can indeed move into industrialization, we decide to design a complete industrial process of nootkatone.

    6.Project and product refinement

    6.1 Factory

    6.1.1 Interview with STAR LAKE

    During the communication, Dr. Cai told us that we should know three questions before going industrialized: what’s our competitiveness, the design of industrial process, and the consideration of the cost. When designing a industrial process, we should first be sure that the strain we use is a high-yielding one. The whole process should be controlled precisely with this condition. At the same time, we learned that separation and purification would account for a large proportion of the total cost. This is what we have neglected, and what we need to think over.

    Through this interview, we generally learned about industrialization and what we need to consider before being industrialized, which greatly inspired our later design.

    Fig.14 the visit to STAR LAKE

    6.1.3 Interview with Pro.Tian

    In order to design a suitable separation and purification process of nootkatone, we consulted Professor Tian, who is more specialized in this field. He gave us some hints on the design of separation and purification process:
    (1). The method that can remove the most impurities is preferred
    (2). Save the most time-consuming and expensive method for last
    After consulting the literature and briefly discussing with professor Tian, we came up with the following general process.

    Fig.16 the interview with professor Tian

    Fig.17 Preliminary separation and purification process

    6.1.3Interview with QR Pharmaceuticals

    To further complete our design, we interviewed QR pharmaceuticals, a hi-tech enterprise which is trying the industrialization of nootkatone. During the communication, they were very interested in our project and gave us several advises. They told us that the most annoying problem of separation and purification is volatile impurities which have similar molecular weight with nootkatone. These impurities will be a difficulty if we want to improve the purity. At the same time, we learned an important point in this interview: increasing the yield of fermentation will bring greater burden to separation and purification, such as introducing other impurities or Increasing impurity content. Therefore, when designing fermentation process, we also need to consider the problems that may occur during separation and purification. Whether existing equipment can meet the purity requirements of separation and purification also should be considered.

    6.1.4 The next steps

    After searching for the national standard of mosquito repellent, we found that the methanol content in mosquito repellent is limited (≤2000mg/kg). So we consulted Professor Tian again on this issue. He indicated that methanol and acetonitrile were the only two mobile phases that could be used in reverse column purification, but acetonitrile was more toxic and cost higher, so the purification method we designed for the last step was wrong, and we needed to revise the purification method for the last step. Since the impact of volatile impurities on separation and purification was specifically mentioned in the interview with QR pharmaceuticals, we also consulted Professor Tian on this part of the content. He mentioned that for volatile impurities with similar molecular weight, solid phase extraction or molecular distillation can be used to further purify the product. However, he suggested that we should consider whether we really need further purification for the sake of cost and purity.

    Learning from the previous experience, we preferred molecular distillation, which does not add additional substances, as our purification method. We also asked QR pharmaceuticals about this, and they also said that they would prefer molecular distillation.

    Therefore, our separation and purification process is modified as follows:

    Fig.18 Modified isolation and purification process

    6.2 Street interviews

    Currently, many people still insist that plant-derived is safe and those involving chemical ingredients and GMOs are harmful, even though this is not the case. In order to further understand and confirm consumers' views on microbial production methods, we conducted interviews on the streets of Guangzhou. For parents with children who need to look after their children's safety, we chose to conduct the interviews in a situation where they could look after their children while answering our questions, such as a children's playground, in order not to cause them too much trouble. The main questions were not only the acceptance of microbial production methods, but also the preference and reasons for whether the mosquito repellent ingredient was a plant extract or a known compound.

    The data showed that the percentage of parents with children who did not accept microbial production methods was much higher than the percentage of others who did not accept microbial production methods, indicating that they would be more cautious in their choice. In the choice of primary ingredient preference, the number of those who chose plant extracts was much higher than those who chose known compounds. This is despite the fact that plant extracts are not necessarily safe, but rather known compounds with stable production processes have a higher guarantee of safety. So we carried out the production of relevant science passages and made more people aware of the actual situation through the public release.

    Fig.19 Acceptance to genetically modified products

    Fig.20 Main component preference

    In addition to this, they also put forward some needs, such as the acceptance of microbial production methods presupposes that the authorities certify the maturity and safety of GM technology, and the masses need to have the right to know. Relevant institutions need to publish the public use, including the assessment of effectiveness and allergenicity, so that the public can choose based on the use of big data. For children, research and development institutions also need to consider whether these substances will produce relevant hazards when breathed in and whether there will be cumulative toxic effects from long-term use. We also asked the kids and they all liked the citrus flavor that Nootkatone has!

    6.3 Guangdong Center for Disease Control and Prevention (2/2)

    In addition to enhancing the personal protection of the general public against mosquito-borne diseases at the main end-use of the product, we also wanted to make a small contribution to solving the problem of insecticide resistance and the widespread spread of mosquito-borne infectious diseases. Therefore, we interviewed the Guangdong Center for Disease Control and Prevention again, hoping to understand the conditions needed for the widespread application of Nootkatone.

    In the first exchange with the Guangdong Provincial Center for Disease Control and Prevention, we learned that the current large-scale eradication will use chemical insecticides, and the round grapefruit ketone also belongs to this range. So, we discussed the issue of chemical insecticides in depth.

    Through the conversation with the expert, we learned that the selection criteria of chemical insecticides for large-scale extermination are, firstly, to comply with the audit by the Ministry of Agriculture, and secondly, not to be on the national list of banned insecticidal drugs. As well as in the public in a wide range of promotion mainly depends on the choice of the market. In order to better popularize mosquito-borne diseases and the active ingredients of repellents to the public, we introduced our current popularization methods to the expert, and she expressed her affirmation of our choice of popularization methods and suggested us to consult the Guangdong Health Education and Publicity Center, as well as to increase our influence by participating in the popularization video or pushing contest.

    6.4 Standard and policy

    After achieving an increase in production and designing the subsequent industrialization process, we realized that if we want to achieve the goal of making a safe mosquito repellent, our project must move from raw material to product in the future. Before any product is put into production in our country, it needs to be registered and licensed by the relevant government departments. Therefore, a preliminary mimetic evaluation of our target product, nootkatone, is also necessary at this stage.

    Some questions arose during our mock assessment.Based on the information available for now, we expect that mosquito repellent prepared from nootkatone will not pose risks to vulnerable populations as reported abroad. However, each country has different testing standards. Since we hope to use experimental data to show the safety of our products for children and others in the future, we would like to know more about the toxicological tests, like whether there are tests to verify the use of pesticides for vulnerable people and whether there are standards or concentration limits in China.

    Since the content and process of approval are available on the government website of China, we have consulted relevant materials and government documents. In a search of the data, we learned that it will require the applicant to carry out animal toxicology test of the product in the testing institution to reflect how much risk it may pose to the user of the product in our country.Perhaps due to ethical and moral constraints, it does not have repellent standards for special populations in our country and the standards for toxicological testing will also not have special requirements for testing. However, the data obtained from the tests will be entered into different models to assess the risk of use for each population under the guidance of Pesticide Health Risk Assessment Report in our country.

    The Health Risk Assessment Model for Repellents takes into account the increased risk of accidental ingestion by children's behavior to assess whether the product can be used by children. If the risk coefficient of the product exceeds the standard in the evaluation result of children, the standard must not be used by children in the product description.We also note that some online arguments say that ‘the concentration of an active ingredient in a safety repellent for young children must be below a certain concentration’, which are actually one-sided.

    The safety test is to test the entire product, not just a sample of different concentrations of active ingredients. Impurities in the product and the selection of appropriate co-solvents and other added substances may also pose risks to users.And different production processes for the production of household pesticides will be classified according to different characteristics, each corresponding to the different toxicological testing.

    After analyzing relevant legal documents and materials, we have a clearer understanding of the legal process from raw materials to products. We also realize the purpose and effect of the steps involved, such as testing for health and safety risks.This provides a very valuable reference and solution to our subsequent project implementation.

    6.5 Product Design

    There are many things to consider from raw materials to products. By searching the relevant information of the government affairs website, we have a general understanding of the registration and approval process for nootkatone as a new pesticide. We hope to design the final product to obtain approval with as little useless work as possible, so we think we should pay attention to the following points when designing the product.

    6.5.1 Detect impurities in raw materials and modify the project accordingly

    During the interview with QR pharmaceuticals, we learned that increasing the yield of the target substance may also increase the production of impurities at the same time. When the project goes to fermentation and purification separation in the future, we need to understand what impurities are difficult to separate in the purification process designed in the fermentation process. If the requirements of the national standard GB24330-2020 are not met, the purification process will be designed accordingly or the engineered strain’ synthetic bypass will be cut off accordingly. In addition, it will also be necessary to test the rest of the impurities clearly and indicate in the health risk report of the pesticide registration.

    6.5.2 Selection of ingredients such as co-solvents in the product system

    We want the safety of our products to be a major advantage in attracting consumers. In the process of searching for policy information , we learned that it is more important to consider the overall product than the concentration of the active ingredient. Therefore, flammable and dangerous substances such as ethanol should account for less proportion in the formula we design. Consider alternatives if there are better alternatives at similar prices. It should be noted that changing organic solvents may also affect the volatility of the product, which indirectly affects the mosquito repellent effect. According to the national standard GB24330-2020, the effective time of repellent clear chemical composition should be longer than 4 hours at least. It requires us to balance safety and efficacy when designing formulations. And we should not use ingredients which are explicitly prohibited in laws and regulations. REGULATIONS ON THECONTROL OF AGRICULTURAL CHEM specifies that highly toxic and highly toxic pesticides shall not be used to control sanitary pests. In addition, active ingredients with low toxicity but high risk are also banned, like Fenobucarb, Sodium pentachlorophenol, chlorofluorocarbon, bis(2,3,3,3-tetrachloropropyl) ether and so on.

    In animal studies, nootkatone has been shown to be less likely to be toxic to humans. However, nootkatone has a certain degree of phytotoxicity, which can cause chlorosis or even necrosis of plant leaves. After searching the literature, we found that these risks and limitations can be greatly improved by encapsulating nootkatone. You can see more about it in Proposed Implementation.

    6.6 Industrialization

    In order to further promote nootkatone to market, we need to have a complete industrial system after having a complete industrial process. Therefore, we plan to further promote the industrialization of nootkatone.

    Fig.21 Preliminary ideas for the industrialization of nootkatone

    First of all, we need to understand what industrialization is. After research in various aspects, we understand that industrialization refers to a certain market economy system, industry demand-oriented, to achieve benefits as the goal of forming a series and branding of business methods and organizational methods. If we want to complete industrialization, we need to do a good job of processing first, and then to do a good job of management.

    The first issue of industrial production is raw materials. The raw material of this project is mainly fermentation raw materials, fermentation raw materials without a specific supply manufacturers, that is, many raw material sellers to choose from, so after the selection of the plant site, followed by consideration of transportation and storage problems, the best is to choose local materials to reduce the cost of raw materials. At the same time, considering the market demand and entrepreneurial ecological excellence of the region, it can not only promote the development of the supply of raw materials in the region where the plant is built, but also to better promote the diversification of the local industry. It can prevent product competition in the process of homogenization to deter the development of new products. Another major factor is temperature, and humidity changes. The temperature is too high or too low will increase the cost of temperature control, and air humidity to a certain extent will affect the content of microorganisms in the air, too high humidity may bring a greater probability and higher frequency of microbial contamination.

    The waste disposal is mainly the treatment of fermentation residual bacteria and waste water. In addition to the cost of treatment, we also need to take the environmental friendliness into account . in China, there are numerous companies that are forced to stop production because they do not follow the principles of pollution treatment in the fermentation industry. Although the cost of pollutant treatment is not low, following national regulations, establishing a sense of corporate responsibility and caring for the ecological environment is conducive to brand influence, while striving to set industry benchmarks and promoting the development and improvement of industry standards for pollutant treatment. Likewise, it has a positive effect on the development of local pollutant treatment plants. Overall, the more companies that follow industrial emissions treatment standards in the region, the better it will be for the development of the local pollutant treatment industry. The main reason for the high cost of pollutant treatment today is that treatment methods are not advanced enough. And when more and more pollutant treatment plants compete in the region, it will promote the improvement of pollutant treatment process, promote the research and new application of pollutant treatment process, which in turn reduces the cost of many plants in terms of sewage discharge and other aspects, and then attract more enterprises to the region and promote the diversified development of local industries.

    Energy, equipment selection and labour are also among the factors that need to be considered. Energy and labour are the more important aspects, with energy mainly referring to electrical energy. When selecting the site for our plant, we considered Guangdong and its neighboring provinces and regions, and finally chose Yunnan as the location for our plant. Since the cost of electricity in Yunnan is only 70% of that in Guangdong Province, the fermentation industry needs a large amount of electricity to keep the fermentation media stirred and the various testing and response components working properly, so it is necessary to choose a city with a well-developed electricity supply system. While Guangdong Province has a wider range of resources in terms of R&D talents, Yunnan Province has a lower labor cost compared to Guangdong. Yunnan Province also places a lot of emphasis on the introduction of talent, and offers significant social benefits to quality researchers, which is one of the reasons why more researchers are attracted to Yunnan. In addition, the key point is that the improvement and optimization of the process in one's own factory will stimulate the peers to carry out the improvement as well, forming a healthy competition. This way, we can promote each other and the process as a whole.

    With the most basic primary product, nootkatone, the next step in the process, which is the processing of mosquito repellent, can be carried out. With more and cheaper raw materials, the cost in the production of mosquito repellent can be reduced. Product prices will be reduced and more consumers will choose our product, that is, when the market demand becomes larger, will also promote the further development of the manufacturer. Likewise, this will stimulate healthy competition among mosquito repellent manufacturers and promote the birth of better products. Good products will also bring business opportunities to middlemen, in this process the media will also play a very key role: to make consumers more aware of our products. Of course, consumers will also give us some feedback on our products, which will help us to further improve our products.In addition to the factors above, a manufacturing license from the government department is also necessary to prove the safety of our products. Because the general public does not necessarily understand what are these specific ingredients, having a manufacturing license issued by the state is the best indication of safety.

    Of course, our product, nootkatone, can also produce not only mosquito repellent. Increasing production of nootkatone in addition to the repellent industry, can also to promote the development of other aspects and even some currently still unexplored applications.

    The production is one side of the industrialization, the market demand for the product is another. Because the consumer demand for the product can drive the production, and the product nootkatone is mainly used in the production of mosquito repellent.According to the preliminary market research mosquito repellent, most consumers demand for mosquito repellent are concentrated in the summer, that is, the main revenue time of mosquito repellent roughly in May to August. Most consumers demand for mosquito repellent is not so much, once a year or half a year to buy, and this product in order to occupy a certain market share need to find ways to improve product awareness in a short period of time. For this reason, we specifically interviewed the Guangdong Biotechnology Industrialization Promotion Association, they pointed out that before and during the sale of the product, we can carry out science popularization activities with the community as a unit, and launch the trial set of this product. At the same time, using our own advantages as a student of SCUT may be more able to attract consumers to try our products, which also coincides with us. This also coincided with our previous idea and further validated the rationality of our envisioned promotion channel. In addition, we also shared with them our vision in the early stage of the business, trying to enter the food market to continue to get a certain amount of profit to support the later research and development and publicity costs, they pointed out that whether it is the food industry or the mosquito repellent industry, are fast-moving consumer goods, in the early stage need to make efforts on brand promotion, to create a good product reputation is the basis for sales.

    7. Next steps

    ...

    ....

    1. Determine the chassis

    After determining to increase Nootkatone by synthetic biology, we started to think about which chassis cells to choose. By reviewing reviews and research articles on biosynthetic terpenoids, we compared the advantages and disadvantages of several "factories" in detail, and finally determined our factory -- Saccharomyces cerevisiae.

    2. Background introduction

    Studies have shown that S. cerevisiae has the Mevalonate pathway (MVA). Valencene synthase (VS) catalyzes the synthesis of Valencene from MVA metabolite FPP. Then Valencene can be oxidized by HPO, CPR and ADH to synthesize Nootkatone. Our PI is Professor Shuang Li from South China University of Technology. Her research group has at least five years' research foundation in the biosynthesis of Nootkatone. The previous research of our group has successfully improved its synthesis in S. cerevisiae cells by enhancing MVA pathway and knocking out or weakening FPP pathway. However, the current maximum yield has room for further improvement and is far from meeting the demand for Nootkatone, a high value-added product.

    3. Choose inducible promoters

    In order to come up with strategies to further increase production, we contacted BIOSYNTHETICA, an emerging enterprise in biological manufacturing. In the communication with the experts, we learned some important information. For the cell factory we constructed to produce Nootkatone, the allogenic metabolic pathways we added would compete with the cell's own metabolism for limited energy and substances in the cell growth stage, while the later stage is an important period for the production of target products. If we use the inducible promoter, we can induce the glucose metabolic flow to acetyl-CoA as much as possible, and let acetyl-CoA be used for the growth of cell in the early stage, and the production of target product in the later stage. Therefore, the expression level of related genes at the later stage of fermentation plays a key role in product accumulation. If the gene does not maintain a high expression level at the later stage, the golden moment of mass accumulation of target products may be missed.

    Excitingly, this has also been confirmed in our literature research. In our in-depth understanding of chassis cells, we learned that the fermentation of S. cerevisiae has the "Diauxic shift", and most of the natural promoters of S. cerevisiae are associated with Glycolytic genes. The difference in the expression level between the pre-fermentation and post-fermentation stages will greatly reduce the efficiency of our cell factories.

    4. Promoter engineering in Saccharomyces cerevisiae

    Then, we discussed these exciting gains with our PI. PI mentioned that when the strong promoter TEF1 commonly used in yeast was used to express Valencene synthase, the yield of Valencene was not high, which was probably related to the Diauxic Shift and Crabtree effect we mentioned. In addition, PI also told us that promoter libraries with a wide dynamic range are of great help for fine regulation in metabolic regulation. Therefore, we focused on the promoter modification of S. cerevisiae.

    5. Choosing inducers

    We started the painstaking literature research. Although it was hard, it was also fruitful. We were inspired by a 2019 paper in ACS Synthetic Biology in which Arun S. Rajkumar et al, based on the "Diauxic shift" of S. cerevisiae, demonstrated a reversal of regulatory function in two glycolytic yeast promoters by replacing glycolytic regulatory elements with ones induced by the diauxic shift to satisfy the need of alternate carbon sources or a need to decouple growth from production. We then applied similar ideas to our wet lab design. According to our production requirements for cell factories, we need to find a trigger to activate gene expression at the ethanol utilization stage, so as to maintain high intensity. Like Arun S. Rajkumar et al, diauxic Shift can be used as a good trigger to make promoters with inductivity without the need for additional inducers to interfere with cell growth.

    6. Searching for promoters

    In the promoter search phase, our goal is to find a batch of yeast promoters with potential and screen promoters for later transformation. At the beginning, our idea was to screen promoters in other types of yeast to obtain greater possibilities. However, through communication with experts from BIOSYNTHETICA, we learned that the replacement of regulatory elements across subspecies was highly unstable, and it was not recommended to consider such a scheme in the early stage of the project. So we limited our search to S. cerevisiae finally.

    7. Molecular cloning experiment

    In wet lab, molecular cloning experiment was a big problem for us due to our lack of experience. At first, we used enzyme digestion and ligating method to construct promoter-VS-SAG1T expression cassette, but PSOL4 could not connect to the vector due to sequence specificity. After consulting with our advisor, we switched to homologous recombination, which is a seamless new method that can avoid some of the sequence specificity problems to a certain extent and quickly solved this problem we met.

    Fermentation Visualized Simulation

    Initially, we wanted to use a set of ODEs (ordinary differential equations) to describe the growth of production bacteria such as yeast in a fermenter, with biomass, substrate mass, product mass, and various environmental parameters as variables, and to use the equations to describe the changes of these variables in the fermenter over time.

    However, in the process of reviewing the literature, we learned that fermentation is mathematically nonlinear, large lag, multivariate, having multiple inputs and outputs, and strongly coupled. Thus, some researchers believe [1] that classical systems theory can hardly establish a suitable model for complex fermentation processes, and it is very difficult to establish an accurate mathematical model.

    1.STARLAKE: From simulation to education

    After recognizing the difficulties, we still wanted to try to build a mathematical model that could simulate the process of the fermenter system over time, and since we could not take into account all the factors that could have an impact, we decided to consult a modern fermentation company, Star Lake Technology, to investigate the most important parameters that fermentation plants are most concerned about in the actual fermentation process, including biomass, substrate, product volume, temperature, pH, stirring force, dissolved oxygen. Therefore, we decided to select only these 7 parameters for modeling. In addition, since the actual fermentation process could not be accurately simulated, it could not be applied to predict the product yield and residual sugar during the fermentation process to provide practical help to the staff in selecting the tank change point. Therefore, we decided to use this mathematical model for educational purposes, both for students of bioengineering related majors to understand the mathematical modeling of fermentation kinetics and for non-specialists to understand more about the changes in parameters and the fermentation process as the bacteria growing, the variation of parameters and the agitation in the fermenter, using the visualization and reproduction tool "meta-cellular automata" to simulate the bacterium and the fermentation process in the fermenter.

    2. Interview with Pro. Zheng: Parameter acquisition

    In attempting mathematical modeling, we encountered a problem that modeling groups typically encounter, which is the difficulty of finding parameters. The literature and reference books provide classical values for only a small fraction of the parameters, which is of course inaccurate. More serious is that more parameters are not yet known, and the amount of experimental data done by our wet lab group could not fit the parameters well, especially in this multivariate mathematical model.

    With this question in mind, we went to consult with Prof. Zheng, who is familiar with fermentation engineering in the college, hoping to get some advice from him on parameter acquisition. The professor pointed out that there is no equation that can describe the whole fermentation process, and the parameters of the fermentation process in the plant and laboratory are usually obtained directly from the pH curve with time using detection tools such as pH electrodes. His suggestion for parameter acquisition is to look for collaboration from the subject or other laboratories, or even to fit the parameters to fermentation process data already available in online databases. But the need to model is not great, because the model is not destined to be fitted accurately, but only made schematic, and the actual industrial production of fermenters is on the tonnage, several orders of magnitude different from the laboratory shake flask fermentation, the scaling up process is not simply equal scale, the situation will be very variable. For example, the ordinary screw stirring paddles in industrial fermenters are unable to provide sufficient stirring force, and baffles are added at the tank walls inside the tank to create turbulence in the fluid field and increase the amount of dissolved oxygen. Turbulence is a complex process that cannot be described by mathematical models and is completely different from the laboratory situation. Besides, mathematical modeling is rarely used in domestic fermentation plants at present. At this point we decided to make the model in the direction of educational science only, and the development focus was not on the accuracy of prediction, but on showing the mathematical thinking in fermentation engineering.

    3. QR Pharmaceuticals: More industrial

    Realizing that we did not know enough about the practical application of fermentation engineering in industry, we set out again to interview the production department of QR Pharmaceuticals. We learned a lot! We learned that the temperature control in the fermentation process, generally need to be artificially warmed up in the early stage to enhance the viability of the bacteria, after the biomass reaches a certain level, the bacteria themselves produce enough biological heat to make the temperature reach the optimal temperature range, and then after a period of time may also need to cool down, depending on the external environment. If the temperature outside is up to 40 degrees, seriously exceed the optimal range of bacteria about 32 degrees, you need to increase the flow of cold water to manually cool down. More importantly, dissolved oxygen is not measured as an absolute value in industrial production, but as a relative value measured for each batch of bacteria before production, which varies greatly from species to species, so we dropped the inclusion of dissolved oxygen in the reference factor.

    4. Dimensional change: From 3D to 2D

    We wrote the set of equations as a MATLAB executable code file and adjusted the parameters according to the reference and experimental data to make it conform as much as possible to the classical process and to favor our own wet experimental data in terms of values. And the set of equations was put into each cell of the meta-cellular automaton, specifically the input and output values at each time point were made into a 2D meta-cellular automaton for the final visualization. We also tried to build a 3D tuple automaton, but due to the poor visualization of the finished product, we chose to present the finished product in 2D again.

    5. Program optimization and future prospects

    In MATLAB, users can easily adjust the set initial parameters, such as the upper and lower limits of the optimal range of temperature, the upper and lower limits of the optimal range of pH, the number of batches of nutrients added in batches and the amount added each time, setting the stirring force and the speed of stirring force rise as well as setting the amount of initial strain input. However, since yeast is not greatly affected by the stirring force, changing the stirring force produces insignificant changes. After setting the parameters, the user can observe the changes brought about by that parameter condition.

    We first conducted a small-scale trial of the program to discover possible bugs. The main targets of the small-scale trial were college students in bioengineering, who proposed it as a combination of classical models and thought it quite interesting to learn. However, because the program is in MATLAB, it is rather inconvenient for those who have not downloaded the software, so we were suggested to change it to a cell phone WeChat applet form. Unfortunately, due to the time limitation, the applet is still under development, and we expect that it will be developed successfully in the future and improve the understanding of the fermentation process for more people.

    3.2 Guangdong Center for Disease Control and Prevention (1/2)

    In the process of experts introducing the basic situation of mosquito-borne diseases in Guangdong Province, we learned that probably due to the warming climate, the distribution range of Aedes aegypti mosquitoes shifted northward, and dengue fever, which should be mainly produced in the south of China, broke out in Shandong in 17 years. This made us realize that mosquito-related science popularization needs to be carried out not only in the south but also in the north, so we cooperated with Jilin China, who are also preparing to go into the field of mosquito repellent, to carry out the promotion of science popularization push so that more people in the north can see our science popularization content.

    4.2 Forestry workers

    People who often need to work outdoors especially in places with more plants are also the people we need to consider. Therefore, we cooperated with Jilin China and consulted some technicians and front-line workers from Xingan County Ganjiang Chinese Herbal Medicine Co. We asked them the main way to get knowledge about mosquito-borne infectious diseases, and they said they mainly through short video software such as Tiktok. This provided us with some suggestions on the selection of media for popularizing related knowledge.

    6.2 Street interviews

    In order to further understand and corroborate consumers' views on microbial production methods, we conducted interviews on the streets of Guangzhou. The main questions were not only the acceptance of microbial production methods, but also the preference and reasons for whether the mosquito repellent ingredients were plant extracts or known compounds.

    The data showed that the percentage of parents with children who did not accept microbial production methods was much higher than the percentage of others who did not accept microbial production methods, indicating that they would be more cautious in their choice. In the choice of primary ingredient preference, the number of those who chose plant extracts was much higher than those who chose known compounds. This is despite the fact that plant extracts are not necessarily safe, but rather known compounds with stable production processes have a higher guarantee of safety. Therefore, we carried out the production of relevant science tweets and made more people aware of the actual situation through the public release.

    6.3 Guangdong Disease Control and Prevention Center (2/2).

    In order to better popularize science related to mosquito-borne infectious diseases and the active ingredients of mosquito repellent to the public, we introduced our current science popularization approach to the expert. And she expressed her affirmation of our choice of science popularization approach and suggested us to consult the Guangdong Provincial Health Education and Publicity Center as well as to increase our influence by participating in a contest of science popularization videos or tweets.

    6.4 Standards and policies.

    According to information on the Internet, people are concerned that mosquito repellents that are pesticides are not safe enough to be used safely on children. But in fact, in the repellent safety assessment has taken into account the use of children and more rigorous safety risk assessment model. However, only those who need to register pesticides will be aware of this matter. We hope to educate the public about the differences in such assessments so that the public will not have unnecessary concerns when using repellents.

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