Simply Survey

We set up a stand in the school to publicize synthetic biology and our projects: C.A.T. Meanwhile, a simple survey was conducted both on the spot and through the official publicity platform of us team.

Our respondents had different educational backgrounds, which can give us a more reliable analysis result.

We found that 67.76% of the respondents had never heard of the concept of synthetic biology, and many people' s understanding only stayed on the simple level of "creating new individuals and new systems". During face-to-face communication, it was found that there was aa lack of good understanding of the so-called bioethical issues among many people.

This suggested that we should carry out more synthetic biology activities with a wider audience, so that people can understand this subject that it is not far from life. Therefore, we also organized and held a large number of educational activities, such as addressing a lecture with topic of synthetic biology, publishing related articles on the public platform, designing a card-game and so on.

We also surveyed the public's understanding of the cat hair allergies. The results indicated that more than half of the people had never been aware of the cause of the disease or the available treatments.

At the same time, our team also made more introductions to students who were interested in our project. Through discussing and sharing ideas with each other, we achieved the two-way communication.

Synthetic Biology Debate

Synthetic Biology has been hailed by multiple well-known scientific media and think tanks as one of the top ten new technological fields that have changed the world, bringing unprecedented changes to the biotechnology industry. To be or not to be, that is a question. However, in today's world where humans have mastered technologies that can encode life, whether to face life with the mentality of a creator is also a question. Based on the consideration of these ethical issues, we held a debate competition on the theme of "Human and Life" synthetic biology. At the same time, we were fortunate enough to invite the iGEM HUST-China to discuss these ethical issues with us.

In order to better showcase the diverse collision of viewpoints, we chose to present it in the form of a debate competition, with the title "In the current rapid development of synthetic biology technology, people have preliminarily mastered the technology of 'creation'. Can humans face life with the mentality of 'creator' ? ".

During the activity, Huazhong Agricultural University held a positive view, believing that humans can face life with a "creator" mentality. Huazhong University of Science and Technology was the opposite side, believing that humans cannot face life with a "creator" mentality.

Both sides' arguments were well founded and won warm applause from all the audience present.

Everyone has gained a deeper understanding of this issue, starting to delve deeper into the relationship between humans and other lives, and enhancing their understanding of ethical issues by constantly expressing their views.

In addition, after the debate, the students expressed their hope to use such dialectical thinking to assist their future subject learning, and to abide by the ethical code of "respecting life, protecting the environment, protecting personal privacy, fairness and transparency, and social responsibility" in the future experimental process. We believe that in the future, we will all face ethical issues in synthetic biology and even the entire biology more rationally and calmly, and use this as an opportunity to generate broader thinking and discussion, and affecting more people.

Seak Professor Xu for advice

Contribution:

Contribution: We induced the expression of TetR using IPTG to test the function of PtetA, but we found that there was no fluorescence even without induction. Professor Xu suggested that this may be due to the leakage of TetR at low doses, which hinders fluorescence expression.

Implementation:

He gave us two possible approaches to improve the situation:
1, Use a high-copy plasmid to carry TetA promoter and a low-copy plasmid to carry LacO-TetR.
2, Reduce the strength of the RBS downstream of Lac operator and increase the strength of the RBS downstream of TetA promoter.

We accepted the ideas and planned to verify them in the latter experiments.

Collaboration with BNUZH-China

Contribution:

In terms of project design, The former leader of BNUZH-China reminded us to pay attention to the selection of the colonization protein for our engineered bacteria. On collaboration in modeling, we engaged in in-depth discussions on the docking process of the same antigen-antibody pair,and shared insights on protein modeling, model optimization, software selection for protein-protein docking, analysis of docking results, and the feasibility of antibody mutation simulation and validation.

Implementation:

Accepted the advice and spent considerable time researching, confirming the structure and adhesion mechanism of the hsa protein used in the colonization module. We also determined the feasibility of the colonization design and developed corresponding experimental protocols for validation. For antibody modeling, we compared the modeling effect between Swiss Model, Alphafold2 and MOE, and finally chose the MOE because of the special Antibody-Modeling function. Other suggestions all helped our modeling a lot.

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Collaboration with CAU-China

Contribution:

CAU-China had made initial progress in constructing a cellular automata model for mycelial growth, while we planned to establish a cellular automata model for bacterial colonization. CAU-China gave the feasible ideas and suggestions for us on the method for designing model rules and engaged in discussions regarding rule design and software selection.

Implementation:

This greatly aided us in establishing our preliminary cellular automata model. After further research and optimization, we successfully constructed a new and improved cellular automata model using NetLogo as a replacement for Python.

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Communication with Dr. Zhao

Because the most common symptoms of allergic reactions caused by Fel d1 on cat hair are skin diseases such as urticaria and papules. Therefore, team member Xincong Sun went to the dermatology department to inquire about the relevant conditions after obtaining permission.

Dr. Zhao, Deputy Chief Physician of Dermatology at Changle People's Hospital, used quite intuitive data to show us the large proportion of patients with allergies caused by animals. Not only that, pet raising families are more likely to experience allergic reactions caused by animals. Even if the owner and family were not originally allergic to the pet, there is a possibility of allergic reactions under the influence of environmental and other factors. This indicates that our project will have a large audience and significant social value.

Secondly, there are some shortcomings in the current coping strategies. Firstly, the main treatment method is to treat after symptoms appearring, by taking anti allergic drugs such as loratadine and cetirizine to alleviate allergic reactions. Furthermore, Dr. Zhao also pointed out that drug treatment has inevitable side effects, such as dizziness, chest tightness, etc. Incorrect use can even endanger heart function. In addition, some groups using a single anti allergic drug for a long time can develop resistance, leading to a significant decrease in treatment effectiveness. Moreover, due to the widespread and difficult to detect distribution of allergens, allergic reactions have become difficult to prevent and have a certain degree of impact on people's quality of life.

Therefore, it is necessary to search for more efficient and convenient prevention and treatment methods at this stage. After briefly introducing our project and products to Dr. Zhao, he affirmed the feasibility of this project and gave a high evaluation of its value. He believed that in a situation where treatment measures were relatively scarce, a priority over treatment was very rare and valuable. If the product could be launched, it could greatly reduce the number of people with allergies and mitigate the impact of allergies, Dr. Zhao hopes that the team can promote the product to the public as soon as possible.

Communication with Dr. Tian

Another symptom of allergic reactions triggered by the Fel d1 protein in cat hair that should not be ignored is asthma. Therefore, our team member came to the Department of Dermatology for a consultation. As the Chief Physician of the Pulmonary Disease Specialist Clinic, Dr. Tian has a wealth of experience in the clinic and answered our questions patiently and in detail.

Dr. Tian pointed out that although the most common symptoms of allergic reactions are mainly skin reactions and it is not common for them to occur in the respiratory tract, respiratory discomfort caused by allergies should also be taken seriously. Some allergic reaction,including asthma will have a rapid onset in a short period of time, with bronchial spasms leading to coughing, chest tightness, shortness of breath, and respiratory distress, which in severe cases can lead to shock and life-threatening injuries. Therefore, severe patients must carry medicines with them in case of emergency.

At this stage, the common countermeasure for allergy sufferers is to stay away from the allergen and take anti-allergy medication even after an allergic reaction has occurred, and there is no better coping strategy. Moreover, desensitisation does not work well in the respiratory tract and is difficult to treat, so there is no therapy to assist in the treatment, and the only way is to relieve the symptoms is through medication. Dr. Tian also mentioned that although the price of medication has dropped dramatically, making it the most economical and convenient way to treat allergies, the side effects of medication and the adverse reactions of individuals to medication also make medication have some shortcomings.

After briefly introducing our project and product to Dr. Tian, Dr. Tian affirmed the team's project direction and pointed out that drugs need to go a long way from the laboratory to the society, and that we need to ensure the safety of the engineered bacteria, make sure that the chassis organisms we use will not be harmful to human beings and pets, and at the same time we should pay attention to the problem of genetic contamination. Dr. Tian encouraged us to put our products into further experimental validation and production applications!

Wuhan Small Animal Protection Association

We also contacted the Small Animal Protection Society. This is because we expect the programme to benefit the stray cats while helping to prevent allergies caused by domestic cats. We have learned that in order to prevent overpopulation of stray cats, the Small Animal Protection Society will regularly de-sex stray cats in the street.

Therefore, we hope to reach a cooperation with the Small Animal Protection Society, and at the same time when they help to de-sex stray cats, they can plant our engineered bacteria in the mouth of stray cats to help solve the problem of allergy caused by stray cats' hair.

After talking to the person in charge of the project, he thought that if our team could work with the Society for the Protection of Small Animals to rescue stray cats and prevent cat hair allergy caused by stray cats, we could definitely achieve great results.

At the same time, we also see the resistance to the cause of stray animal protection: the influence of traditional concepts, the lack of relevant laws and regulations, and the shortage of rescue funds etc. Members of the Small Animal Protection Society are always in the dark corners of no man's land to send a beam of light to the stray animals who are full of scares and homeless. So we hope that, through our project, we can join with the Small Animal Protection Society to call on the whole society to pay attention to the cause of stray animal protection.

We also hope that through our project, together with the Small Animal Protection Association, we can call on the whole society to pay more attention to the cause of stray animal protection, and send a little light to these caring rescuers, so that they can become an ocean of light and illuminate each other.

POP PET SHOW

In order to understand the needs of our customers deeply and to verify the feasibility of the product's form, we participated in the Wuhan POP PET SHOW, hoping to take this opportunity to connect with more stakeholders. At the initial stage of the project, we hoped to make the product in the form of a chewing toy based on the site of action of the engineering bacteria, and with this initial idea, we communicated with a number of pet product brands at the show.

Through communication, we learnt that cats are more accustomed to licking than gnawing ,due to the influence of their teeth and oral structure, so the form of chewing toys is not feasible. Moreover, many merchants also pointed out that owners have been paying more attention on the overall care of their cats in recent years, so the sales of probiotics have shown unprecedented growth. Therefore, for the above two reasons, as well as the choice of engineering bacteria in the experiment, we decided to make the product in the form of probiotics nutrition gel for cat.

Dialogue with pet supply merchants
- stakeholder value input

In order to let the experimental group have a better understanding of the needs of the market and the concerns of the stakeholders, and for the stakeholder groups to understand the progress and results of the project better, we also interviewed a number of pet supply merchants in both online and offline forms, including big brands such as Reddog, XCPET, Poainta, and so on.

When we proposed our products and their efficacy to the businessmen, they firstly expressed their recognition and affirmation that our products have high value of use and economic value. However, at the same time, they also raised some concerns, such as: the effect and duration of action of the antibody, the selection and cultivation of strains, the safety of the product as well as the relevant production qualifications and inspection reports of the product. At the same time, the final marketing of the product should also meticulously study the corresponding market regulation and laws and regulations to ensure that our product complies with the regulations.

Product Questionnaire

After determining the product form of our project, we issued a questionnaire named "Research on Pet Nutrition Cream Products for Inhibiting Cat Hair Allergy" to the society, and 183 valid responses were received. Next we conducted statistics and analysis on them.

Part I: Background of respondents.

Our respondents have a wide age distribution and diverse educational backgrounds, and 19.28% of them have relevant backgrounds (including biotechnology, product development and product sales), which allows them to give more accurate opinions and suggestions.

In addition, we found a large proportion of people are raising pets, and half of our respondents have cats (22.51%) and dogs (30.3%), which are potential customers of our products. The proportion of 4.92% allergic patients may be lower than the value obtained from the background survey due to the small sample size. However it still indicates that our survey successfully targeted the product demanders.

Part II: Market situation of similar products.

We found that the biggest problem with existing products was the recurrence of allergic symptoms, followed by concerns about the need for repeated treatments and high price. This is exactly the focus of our project. Feeding probiotic nutrition gel for cat once enables our engineered bacteria to colonize and proliferate in the oral cavity, continuously producing neutralizing substances and preventing allergy in the continuous iteration of bacteria. There will likely be a significant reduction in the recurrence of symptoms and the frequency of product use.

Part Ⅲ: Suggestions for our products.

Firstly, the questionnaire survey suggested that we should focus on the research and development to enhance the preventive effect of our products (29.91%). Secondly, product costs should be appropriately controlled (22.37%), as far as possible to keep the price of each treatment below 200RMB, to improve the competitive advantage of the product in the market. Thirdly, we should ensure the safety of both development technology and production process. We specifically designed the designed a double-layer suicide circuit for our engineered bacteria so that it can immediately switch on suicide when exposed to unexpected situations outside the mouth and into the digestive tract, avoiding genetic contamination and harm to the human body. In addition. It also suggests that we should continue to pay attention to production safety of our product in the future.

Stakeholder Analysis

We planned to transform the project design into a product by producing a probiotic cat treat. Stakeholder analysis allows us to plan and establish an equivalent level of communication based on their impact and interest in the project.

Firstly, through extensive brainstorming sessions with the entire HZAU team, we identify all the stakeholders associated with this product. We involve all the stakeholders to understand their values, needs, the impact of the issues, and the potential impact on them. We create a list of individuals and groups who are influenced by our project, including investors, advisors, government, teammates, and even family members, and categorize them according to production, demand, competition, collaboration, and management.

Next, we analyze the identified stakeholders. One simple yet effective stakeholder analysis technique is a stakeholder map. The most renowned model is the Power-Interest matrix. This matrix groups stakeholders based on two variables: power (their ability to influence our project and strategy) and interest (their level of interest in the success of our project). The resulting stakeholder matrix clearly identifies key participants who have the greatest impact on the success of the plan. This map helps to prioritize resources and serves as a basis for communication and engagement plans. (This useful tool originated from researchers at the Tavistock Institute of London in the late 1960s and early 1970s).

This matrix tells us how to communicate with different groups during the project development and production.

● High power, less interested people: Understand their goals in detail and meet their needs as much as possible. Therefore, we try to do this in person through occasional website release or email contact, feedback our ongoing activities to relevant government regulatory organizations, and try to raise their attention.

● High power and highly interested people: Key people, spend the main energy on this group of stakeholders, involve them in the design and decision-making process, and maintain close contact throughout the process. This means that we aim to fully engage these stakeholders through regular meetings and keeping them constantly informed. Discuss with the project designer, the product's producer, and the sponsor about all the choices we have made and all the progress we have booked in the project.

● Low power and less interested people: The least important group, communicate with them through regular channels. We can try to raise its attention. For competitors, we also need to pay attention to their progress of the development and production.

● Low power and highly interested people: We can select some important concerns of our project, and do some attempts that will not have a negative impact on the core of the project. Because they are potential communicators of good reputation. We support them to follow us on social media and keep abreast of our development and production progress. We will also continue to integrate their needs into our design to achieve a win-win situation.

Suggestions By Model

We used NetLogo to create a cellular automata model of colonization, intending to visualize the process by which our engineered bacteria reproduce, colonize and compete with other bacteria in the cat's mouth. When searching the relevant parameters and building the model, we found that only when engineered bacteria can be provided with sufficient energy can they colonize smoothly.

Therefore, the probiotic nutritional gel we designed required redesigning and special attention to ingredients.

It is known that the main function of cat nutrition gel is to provide cats with some nutrients and help cats eliminate hair in their intestines. The composition of hairball control nutrition gel with the brand of Nourse was analyzed as an example.

Carbohydrate is not naturally found in the diet of the carnivorous cat. Nevertheless, pet foods contain considerable amounts of tis ingredient. Canned foods can contain up to 30% carbohydrate, and the greater part of the carbohydrates found in these products is starch. Total apparent digestibility of starch in adult cats was reported as varying from 40 to 100% [1,3]. Some investigations showed that cats are unable to metabolize sugars in large quantities [2-4].

However, current study from L. D. de-Oliveira showed that all sources of starch (cassava flour, brewers rice, corn, sorghum, peas, or lentils) showed satisfactory digestibilities, indicating that they can be used in the formulation of cat food. Therefore we chose starch to power our engineered E. coli[5].

In addition, the starch content in our nutrient gel should be adjusted to meet our needs: First, provide enough energy for the engineered bacteria to colonize and continuously produce antibodies; Second, within the limits of how much starch a cat can digest; Third,make sure it will not produce too much sugar in the mouth to cause the excessive growth of streptococcus mutans, wich may exceeds the capacity limit of the sterilization module of our engineered.

In the future, the specific ratio and composition need to be adjusted after discussing with some professiona lcat researchers, experienced product designers and manufacturing enterprises of nutritional gel.

Reference

[1]Morris JG, Trudell J, Pencovic T. Carbohydrate digestion by the domestic cat (Felis catus). Br J Nutr. 1977 May;37(3):365-73.

[2]Kienzle, E. Carbohydrate metabolism of the cat. 1. Activity of amylase in the gastrointestinal tract of the cat. Animal Physiololgy and Animal Nutrition.1993;69:92-101.

[3]Kienzle, E. Effect of carbohydrates on digestion in the cat. Nutr. 1994b;124(Suppl. 12):2568S-2571S.

[4]Washizu T, Tanaka A, Sako T, Washizu M, Arai T. Comparison of the activities of enzymes related to glycolysis and gluconeogenesis in the liver of dogs and cats. Res Vet Sci. 1999 Oct;67(2):205-6.

[5]de-Oliveira LD, Carciofi AC, Oliveira MC, Vasconcellos RS, Bazolli RS, Pereira GT, Prada F. Effects of six carbohydrate sources on diet digestibility and postprandial glucose and insulin responses in cats. J Anim Sci. 2008 Sep;86(9):2237-46.