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Human Practices

Overview Integrated Human Practice Achievement

Overview

In order to allow our project to better connect with the world, instead of just staying in the laboratory, we have carried out human practice activities, choosing different methods according to different groups of people, and improving the public's understanding of synthetic biology and our project. In this process, get feedback that will help our products to be put into production.

Integrated Human Practice Overview Map

Integrated Human Practice should always involve two-way dialogue - introducing a project to the wider public and getting their response in return. Integrated Human Practice is especially important for our production of 2-FL this year - since our ultimate aim is to commercialize our product and making it handy for people , no matter it be used in dietary supplements and medical foods, or infants formula. we would have to better our product through meticulous planning and adequate conversation with expert. Thus, we devoted a lot of time and resources to contact all sorts of people, asking about their opinion of our project and receiving suggestions and ideas for improvements. Here is a detailed record of all our Integrated HP activities - who we talked with, what they got from us, what we got back from them.

1.Public Opinion

2.Stakeholders’ Views

After the first round of background research, we believe that the public's attention to the content of milk powder and HMOs is not high. After discussion, we believe that the scope of the survey should be narrowed and further attention people related to the sale and purchase of milk powder products. We interviewed stakeholders as baby mothers, milk powder distributors, and child care workers. Their opinions can provide advice on the practical application of our project to production and sales.

a.baby mother

Q1: Do you pay attention to HMO content when buying milk powder?
baby mother: I pay more attention to the brand effect, but at the same time, it needs to be safe enough. At least the baby will not have diarrhea after drinking it. Usually, there is a certain period of adaptation for a new brand of milk powder.But what is HMOs？
Li Xintong: HMO is a kind of breast milk oligosaccharide contained in breast milk, which can help babies improve the resistance of gastrointestinal immune system. It should be suitable for babies with poor immunity.
baby mother : Is it suitable for allergic babies? Should be some babies just born physique easy to allergic, may be more than the mother will care about this. But I don't know much about it.

Q2: Is it acceptable to use yeast to produce HMO in milk powder?
baby mother : I only know that this is very popular (Nestle super high-end Nen Quan Care 5HMO probiotics moderately hydrolyzed hypoallergenic milk powder 270/350g), which is basically premature ah, allergic babies drink this, very expensive.

Q3: Is the price of milk powder containing HMO acceptable in the current market, and the acceptable price range?
baby mother : The price is OK and acceptable.It's about 300/800g, which I can accept. If it's over 400, I can't.

Q4：What ingredients did you pay attention to when you bought milk powder before?
baby mother :Because I'm a big advocate of exclusive breastfeeding when I can.Breast milk is one of the best sources of nutrients for a newborn baby.But if you can't breastfeed exclusively, for example, because of your health or your job.I would certainly choose some milk powder whose ingredients are more similar to breast milk as a substitute for breast milk for babies.I might be paying more attention to his brain development,and the components of things like immunity.

Q5：Do you think this project will be attractive to mothers like you if it is really successful？
baby mother :For a determined breastfeeder, if there is no way to complete breastfeeding due to work or physical reasons, then the best alternative is dairy products that are as close as breastmilk. I think your project will be able to solve a large part of the problem with the busy mother

We interviewed a total of 8 milk powder salesmen and one person was willing to be photographed and two people were willing to be recorded. The following is a summary of the interview.

b.Store Clerk

Q1：After learning about our project, do you think it makes sense to use yeast to produce HMOs? Can it be accepted by consumers?
Store clerk1：The content of HMO is high in breast milk, but not in current milk powder, because it may be difficult to produce. HMO can improve the immunity of babies and contribute to the growth and development of babies. Therefore, I think the production of HMO with yeast will play a positive role in the development of milk powder industry. However, since I don't know much about your project, I don't know whether such production may produce some disadvantages, so I can't be too sure. I think customers who know something about HMOs are acceptable.
Store clerk2：I don't know much about HMOs, and I don't know much about your project, making HMOs with yeast, but IF it's a genetically modified product, I'm not very comfortable with it. You say THAT HMOs are important for infants and toddlers, so it might be nice to be able to produce them in large quantities and add them to milk powder, but I'm not comfortable with it.I don't think it would be acceptable for consumers who don't know much about HMOs.

Q2:Are there any customers who will first consider milk powder with HMO ingredients when buying milk powder?
Store clerk3：When consumer is buying milk powder is more focus on protein content, nutrient content, but most people with price as the reference standard, think that the more expensive the choice, the better, some customers of milk powder have research will try to choose the milk powder containing lactoferrin or HMO, hope to be able to try to restore breast milk composition, but most people don't pay attention to the content of HMO.

Through interviews, we found that a minority of stakeholders have an understanding of HMOs and that this ingredient can improve infant immunity. At that time, when the price was too high, mothers were distressed. However, the current price of dairy products containing HMOs is indeed very high. Through the milk powder store clerks, we have also learned that the production method of HMOs has increased its price.

3.Expert Advise

Jingyu Chen, associate professor and doctoral supervisor
Research field and direction：
1).Environmental response and regulatory mechanism of food-borne pathogens；
2).Synthetic biology and future food manufacturing；
3).Development and utilization of microbial resources of food starters；
4).Molecular mechanism of oxidative stress response in Saccharomyces cerevisiae.

We interviewed Chen Jingyu, Associate Professor of China Agricultural University. The professor answered every question in detail and enthusiastically. Mr. Chen has a lot of experience in the application of yeast in synthetic biology. And he is very familiar with the fermentation engineering of using yeast to produce food, which can guide us to better optimize the project, avoid many production risks in advance, and also have many ways to increase production.We first introduced our project design to Teacher Chen, and then started the discussion.

a.Problem Outline：

Q1: What do you think the project can continue to improve? /What work or supporting measures are actually needed for input? What are the problems?
Q2: We understand that the teacher has a lot of excellent research results in the field of yeast fermentation. What do you think is the advantage of using yeast fermentation to produce HMO by means of synthetic biology? (Compared to direct extraction or chemical synthesis methods)
Q3: In the previous survey, it was found that the relevant consumers are not very accepting of HMO in the production of milk powder using synthetic biology methods. How to optimize our project to make it more safe? (Including pathways involved, fermentation conditions, subsequent product purification and packaging production, etc.)
Q4: We understand that the teacher's research field involves "synthetic biology and future food manufacturing", but the public still has some misconceptions about genetic modification and biosynthesis. As students, can we make a commitment to social responsibility? What efforts are you making? For example, conduct biology science lectures and distribute leaflets for elementary and junior high school students.
Q5: Are there any environmental safety hazards in the project, such as genetic leakage? How to avoid it?
Q6: The experiment is currently undergoing some molecular biology operations. We expect to use three different promoters (GAPDH, TEF1, ADH1) to drive the expression of the three genes FKP, FucT2, and Lac12, and then combine the gene fragments with the promoters The fragments are integrated into the yeast genome with the help of homology arms and then fermented. Is there anything that can be perfected in this design? Are there any areas that require special attention in the later fermentation?

b.About Problem

Teacher Chen mentioned that in the production of food fermentation, the commonly used strains are Saccharomyces cerevisiae and Bacillus, the latter has a good exocrine pathway, and the use of widely used strains is more convenient for project implementation. We also have more reference materials. After understanding the possible drawbacks of E. coli production, Teacher Chen affirmed that we use yeast for production.
Teacher Chen believes that this is the innovation of the project, because fermentation relies on high biomass. For yeast, it grows quickly, has higher productivity, can be cultivated in large quantities, and is affordable. In addition, the promotion of later products is also more favorable, and the public may be more able to accept yeast as a chassis organism for production.Regarding product safety, Teacher Chen mentioned that it must go through a strict safety review in the later stage. At present, the food production industry has a very detailed and very mature safety evaluation system. Our products must undergo safety evaluation and meet the standards before they can be sold normally.
Feedbask: After Teacher Chen’s exchanges, our safety awareness has deepened, we have carefully conducted literature research, clarified the safety assessment process and precautions for subsequent product production, and selected reliable and regular manufacturers for production to alleviate the concerns of stakeholders. The content is also reflected in the proposed implementation. And in the follow-up product promotion and publicity, the safety of yeast and its wide use in food production are emphasized.

c.About Design

Teacher Chen pointed out that in order to obtain the final product, a good cell secretion pathway is needed. After learning that we hope to obtain the final product by crushing the cells, Mr. Chen suggested that we can add a suicide switch to the cells, and at the same time remind us that the activation of suicide enlightenment is rapid, but it needs to be induced. At the same time, cell death does not mean the complete release of the product, because the product still exists in the cell, and it may take a long time to fully obtain the product in the cell.

Feedback: After the exchange, we checked the literature and found that the suicide switch needs to be activated under the guidance of the outside world, and the efficiency of obtaining the product is too low. In addition, this requires constant replenishment of yeast cells during the fermentation process. In the actual fermentation process, the economic benefits are low. At the same time, we discovered a usable protein-CDT2(proof of concept), and applied it in our project.

d.About Fermentation

Mr. Chen is an expert in fermentation. After learning about our project, Mr. Chen reminded us that yeast cannot make good use of lactose, so this is an advantage, but fucose as a raw material is more expensive, and in the actual fermentation process, We need to continuously expand the fermentation conditions and explore the most suitable fermentation conditions step by step through shake flask experiments, from 1-2L to 5-10L, and from ten to one hundred times.

Feedback: This indicates that our fermentation conditions need to be improved, and the most suitable conditions need to be explored, and we hope to improve the fermentation raw materials, such as the use of glycerol, but whether it is feasible or not needs to be verified by later experiments.

How to close the loop between design and desire

1.Silver

a.We conducted interviews with stakeholders and learned that consumers have a high demand for breastmilk substitutes, but due to the high cost of synthesis of breastmilk ingredients, the prices of products on the market are relatively high, which supports our project background.
bWe conducted interviews with stakeholders and communicated with Associate Professor Chen. They affirmed the use of yeast as a chassis organism for biosynthesis, and expressed that it can greatly simplify the downstream process train of product production.
c.We talked to Associate Professor Chen to understand the safety and ethical issues that this technology may involve, and to understand the public’s concerns about the safety of our project by issuing questionnaires.

2.Gold

a.We talked with Associate Professor Chen and found that it is very inefficient to obtain products by crushing cells. By consulting the literature, we chose CDT2 as the product efflux pathway.
b.We talked with Associate Professor Chen and found that too many plasmids would increase the burden of biological metabolism of the chassis and cause problems such as loss. Therefore, we used one plasmid and designed a homology arm to improve genetic stability.
c.Through literature research, interviews and questionnaires with the public, we rationally plan the use of the project and promote it to the market in a safe manner.