Overview

Human Practice and Integrated Human Practice, emphasizing how the world affects us and how we affect the world, act as important links between iGEM competition and our real life. In this year's SHSBNU_China project, our team members connected with people from all walks of life to impact and be impacted. Through 2 field visits, 2 individual interviews, 2 pieces of questionnaires and 4 online communication, we received inspiration, encouragement and suggestions regarding even the smallest experiment details. We also reacted to the feedback, amended our strategies and lab plans and communicated our results with the communities when there is improvement, which also harvest a lot of support.

This part mainly includes the process of our Human Practice and Integrated Human Practice. We divide them into four stages: “Project Idea”, “Project Feasibility”, “Project Design and Modification” and “Project Expand”.

Integrated Human Practice Hightlights

Figure. Among many HP activities, there are a few key suggestions we integrated into our project through the various stages. They greatly helped and shaped our project. You will read about details in the following documentations.

1.How did we find the project idea?

Offline and online questionnaire: primary and middle school students
- We focus on improving youth vision problems through synthetic biology methods.

At a brainstorming meeting long before, our team members mentioned a myopia glasses shop was lining up these days near our school. We were surprised to realize that there was a high proportion of students wearing glasses in our class. In the upper grades, there were almost no students with normal vision. Even a large proportion of the students in the lower grades also became short-sighted early on. To verify our conjecture, we firstly designed a myopia questionnaire for primary and middle school students. In order to get more extensive information, we conducted online and offline surveys jointly. The offline respondents were people in Beijing, and the online survey was conducted all over the country. Based on the data we collected: The number of myopic people is more, accounting for about 75.89%, the number of non-myopic people accounts for about 21.99%, other eyesight problem like hyperopia is less, but also accounts for about 5.67%.
The survey data we obtained verified our prediction that more than three-quarters of the people in China are nearsighted, and vision problems constantly affect people's work efficiency and quality of life. Therefore, the team of SHSBNU_China selected the topic of this year's project, we will focus on improving eyesight through synthetic biology methods.

Figure 1. Surveyed results show 75.89% of the people of our age group has nearsightedness. Only roughly 22% of the young people we surveyed have no vision problems, wearing no glasses of any kind.

Figure 2. Results of multiple choices question surveys, and the answers are not exclusive. Our survey also showed user preferences for treating vision problems. The majority (85.82%) of the users will choose to strengthen vision health with a product of some kind. One respondent can select as many choices as needed, and therefore the total of the pie chart exceeds 100%.

Personal interview: Students and Parents
We thought about a dye that could block blue light.

Furthermore, we selected some students and conducted personal interviews with their parents. We learned that most of the children had been wearing myopia glasses when they were in middle school, and the degree of myopia deepened year by year with the rise of grades. The largest number of people think that electronics affected their vision, other people also talked about genetic factors.
After searching the scientific papers, we found that many electronic screens can emit blue light, which is largely existing in computer monitors, fluorescent lights, mobile phones, digital products, and so on. This kind of blue light increases the number of toxins in the macular region of the eye, which can cause serious damage to our vision. Consequently, we thought about whether there was a substance that could block blue light, and we first thought about a blue dye, and we hope it can absorb blue light for the protection, and also we hope to find a substance that can have protective functions to the cells.

In-person interview: Prof. Geng from Minzu University of China
We decided to make an anthocyanins dye through synthetic biology.

We took our idea and went to Prof. Geng from Minzu University, where she suggested we try anthocyanins, a natural dye found in plants. The color diversity of plants is mainly controlled by anthocyanins, and different types of anthocyanins can give plants different colors.
To meet our demand for blue light protection, Prof. Geng recommended delphinium which has the color blue. At the same time, Prof. Geng introduced to us, anthocyanins can also provide protection against oxidative stress and are the most potent antioxidants discovered up to now, it can truly provide a variety of benefits to the human body.
We were all excited about Prof. Geng's proposal. We decided to make an anthocyanin dye through synthetic biology and use it to protect people’s eyesight.

Figure: SHSBNU-China team members (1st Left Huang, 2nd Left Zhang, 1st Right Zhao) and Prof. Geng (2nd Right) had an in-person interview to introduce anthocyanin from various plant orgins.

2.How did we prove it to be feasible?

2nd Offline and online questionnaire: everyone interested in our project
People know little about anthocyanins but showed positive attitude toward a new product.

To prove the feasibility of our proposal, we first designed a questionnaire to ask people about their understanding and use of anthocyanins, which was also conducted both online and offline at the same time to obtain more data.
We found that anthocyanins were poorly understood by the market and the public, which laid a good foundation for our education campaign.
Some data are recorded as follows:
1) Only about 3.5% people know anthocyanins.
2) The frequency of utilization of anthocyanins is only about 0.71 percent.
3) People use anthocyanins around 0.8% of the time.
4) Only 3.55% of the people were engaged in anthocyanin-related work, and 96.45% of the people were not engaged in any related works.
Despite this, people were very positive about anthocyanins helping to improve eye health, followed by boosting immunity, improving allergies and controlling blood pressure. If we had a product rich in anthocyanins to help people protect their eyes and enhance their eye health, there would be a very large audience and a very large market: 59 percent were willing to buy anthocyanin products, while 41 percent were unwilling to buy related products.

Figure. Only 3.55% People knew something about anthocyanin

Online communication: Dr. Ligong Shao from Beijing Shouer Li Qiao Children's Hospital
Our idea is completely feasible.

In order to get more information from the expert, we reached Dr. Ligong Shao from Beijing Shouer Li Qiao Children's Hospital, who is a Doctor of Ophthalmology and visual development science, postdoctoral of pediatric ophthalmology and optometry medicine. Dr. Shao has made profound and outstanding contributions in the field of ophthalmology, visual development science and ophthalmic medicine in China, and has an important influence, known as a famous expert in pediatric ophthalmology.
In our interview, Dr. Shao told us that anthocyanin is a neurotrophin without any toxic and side effects, which can indeed play a protective role on the eyes, such as auxiliary nutrition, supporting the survival and repair of retinal pigment epithelial cells, as well as assisting the correction of amblyopia, prevention and control of myopia, eye health rehabilitation and so on. At the same time, Dr. Shao affirmed our idea that anthocyanins can be made into tablets, capsules, liquid sprays, plasters, eye patches and eye protection liquid, etc., under the premise of ensuring scientific, standardized and safe operation, our idea is completely feasible.
Therefore, we proved the feasibility of the project and started the project.

Figure, Interview chats with Dr. Ligong Shao assured us feasibility of using anthocyanin on a protecting product.

3. How did we design and modify our project?

In this part, we reviewed the papers to find anthocyanin production pathway in plants, and determined the final sequence by codon optimization. We constructed the vector by means of Gibson Cloning, but we encountered many problems in the experimental process. We found a variety of experts to help us and responded to each one.

Field visits: Dr. TianZe Zhu from Beijing Zeno Technology Development Co. LTD We followed his advice and studied the best reaction duration, which we later found to be around 16 hours to express the enzyme.

Our first problem is to increase the expression of our enzymes. We contacted Dr. Zhu, who is a former IGEMer, and is now an entrepreneur who aims at extracting biological pigments for dyes using synthetic biology. He took us to visit his laboratory and introduced various wet lab departments. The departments include ordinary molecular biology labs and also uncommon production and characterization lab. It was a great experience for us to learn about the real-world production of a substance using synthetic biology. We presented our project and had a Q&A discussion with him.
In the discussion, Dr. Zhu patiently asked about the experiment details in our project design to help us analyze how to improve the enzyme production. Dr. Zhu told us the expression level is relatively high when the OD level is higher in the bacteria culture, which means we could adjust the reaction time to gain a better expression. There is no magic number and we have to carry out experiments to study the express at different reaction durations. We took this important advice back to our lab and did a time-course experiment, and find the best time to express is around 16 hours. You can read more about the experiment results on our results page.

2nd Field visits: Prof. Geng from Minzu University of China
1) We designed the experiment to detect the intermediate product first.
2) We adjust the pH to see if there's a color change. 3) We learned a tool to analyze SDS-Page numerically.

The second problem we had after increasing the expression of the enzyme was that instead of the blue anthocyanin product, we saw the pink solution. Therefore, we contacted Prof. Geng from Minzu University of China. Prof. Geng affirmed the progress of our project and put forward the following suggestions regarding the problems of our current experiment:
1) First, whether some important intermediates can be tested.
Based on this, we detected the intermediate product naringenin in the pathway. We searched the papers and found a naringenin detector, which can express a protein to specifically bind to naringenin. The compound will then activate the expression of a reporter. We designed the experiment to detect the intermediate product first. Refer to our Results page for more on this part of the experiments.

Figure. Expression of the reporter enzyme with addition of 50 and 100 nM naringenin.

2) Second, Professor Geng suggested that anthocyanins may be affected by pH. Therefore we carried out this experiment to change the pH to see if there's a color change. See in our Results page for more details.

3) We having been actively looking for ways to quantitatively analyze SDS-Page results. We already played with Photoshop to draw a square around the protein band and find out the value from Photoshop. Prof. Geng praised our efforts and creativeness, and recommended a professional tool ImageJ to us. We learned a great deal on this, and you can refer to our Measurement page for more.

Online communication: Dr. Ding Dan
A new product using delphinidin is proposed.

We communicated with Dr. Ding Dan from Good Vision Tech and asked him for advice. He works as a R&D department staff on eye pads. With rich product development and design experience, Dr. Ding Dan confirmed our project to be feasible in theory and effect. We were advised to use delphinidin to replace the medicinal ingredients in our daily eye pads. Dr. Ding Dan expressed interests to help us communicate with Good Vision company to solve the problem of making and marketing delphinidin eye pads although it will take a long time about 3-5 years.

Figure. Customers usually use eye pads for skin care. Good Vision developed a product that contained authorized medicinal compound in the padding materials to help eye protections.

4.How did we expand our project?

In this part, we mainly bring our experimental data to the experts we had interviewed before and asked for more advice. Fortunately, all the experts agree that our results are meaningful and could lead to great advances in eyesight protection. At the same time, they proposed new directions for us to explore. We improved our project design based on their feedbacks through adding safety considerations to the future work and planning extra investigation for applications.

2nd Online communication: Dr. Ligong Shao from Beijing Shouer Li Qiao Children's Hospital
We realized it is important to do more cellular biology experiments in the future for safety reasons.

In this time’s interview, Dr. Shao's major focused on the irritation and tissue reaction of anthocyanin. He told us although anthocyanin is a nontoxic and harmless product, there may still be some people shows allergic to it. As a result, we realized it is critical to carry out more cellular biology experiments to figure out the dosage of our proposal before the proposal could become a product. For safety reasons, we did not have the opportunity, the necessary experiment conditions, and the proper training to conduct cellular biology experiments. We emphasized the importance of this step in our future work and integrate this advice into our Proposed Implementation.

2nd Field visits: Dr. TianZe Zhu from Beijing Zeno Technology Development Co. LTD His suggestions helped us plan to investigate color fastness and how to dye contact lenses.

This time, with a successful production of the desired anthocyanin, we took our results to look for Dr. Zhu’s advice on how to make a better product. He affirmed our preliminary results and pointed out that biosynthetic dyes have certain prospects and advantages over traditional methods, especially in the aspect environmental protection. Mass scale synthesis of plant-based dyes is worth exploring for technical and economical reasons, and our efforts are important. His comments improve our confidence in the project.
At the same time, he suggested that we could explore some problems we might confront when we take our next to make a product in real life. He put forth a new problem of color fastness, which means the dye on the product could fade under various conditions, such as washing, illumination and friction. When we make a colored contact lenses with the anthocyanin for eye protection, we have to consider if the protection substance would quickly lose from the lenses. Many contact lenses are disposable and will be changed (trashed) daily. It is not very important for disposable things to hold the color for long, but it is really worth thinking about when it comes to long-term usage. A related issue is how much time it would take to properly dye the lenses. We were excited to learn these new aspects and we made a plan to observe and test dyeing and de-coloring. Please visit our results page for the dyeing and de-coloring experiments.

Online communication:Zu Yong from General Manager of Prokaryotic Biotechnology (Shanghai) Co., Ltd.,

Zu Yong, male, 2017's doctoral graduate in biochemistry and molecular science from the School of Pharmacy of East China University of Technology, General Manager of Prokaryotic Biotechnology (Shanghai) Co., Ltd., and currently self founded a digital medical technology enterprise.
On September 20, 2022, we, together with iBowu_China, interviewed Zu Yong, General Manager of Prokaryotic Biotechnology (Shanghai) Co., Ltd.
We introduced our project to Professor Zu Yong and asked the following questions:

1.In what possible way can we maximize our productivity, and the expressiveness of the proteins.
2.In real-world production we dyed the contact lenses, is there anything that we need to pay attention to on safety aspects?

Answer:
1.The concentration of coumaric acid oleoresin substrate is not enough.
2.The intermediate product, the outcome quantities and reaction rates of each enzyme need separate testing.
3.For temperature, you can try personalizing in accordance to the condition of each enzyme, for instance the initial temperature, how long is this temperature kept, what temperature will later be set, for how long.
4.The safety of contact lenses indeed requires consideration. We produced contact lenses dyeing only to prove the feasibility of the product, and the craftsmanship in the industrial production in reality, but its toxicity and safety aspects still need consideration.
5.Since the eyepatch can be made, then more storytelling is needed, like what the principle is, how the effects look like.
6.Later on we will do some improvements on the safety descriptions: we are now testing the effect of dyeing on the daily-thrown contact lenses. In the case of the formal production, we need to strictly follow the production safety protocols, which, in China, belongs to Category III of the Regulation on the Supervision and Administration of Medical Devices and the Specification for the Production Quality Management of Medical Devices, we will verify further as a future plan.

An interview with Prof. Qingli Wang

We interviewed Professor Wang Qingli, director of the Toxicology Department of the Pharmacology Department of the State Food and Drug Administration. He has rich experiences in pharmacological reviews and provided help to the safety of our contact lenses-dyeing experiment.
Q: Is there any way to verify the safety of our contact lenses and eyepatches?
A: Contact lenses using delphinidin are completely feasible. However, its safety aspect needs further investigation. As a listed product, the safety of delphinidin eyepatches is much higher than delphinidin dyeing. Specifically, when it comes to the safety verification, the first thing is to experiment with white mice. Add delphinidin solution to the mice’s retina to see if the solution can cause side effects on the eyes, since delphinidin’s toxicity may attack the mice’s optic nerves. The solution can also be added dropwise on the mice’s skin to verify allergy reactions. For the supervisors, this experiment shouldn’t be too hard.
Q: Is there anything we can do to improve safety?
A: There aren’t many problems in other areas of the experiment, but be aware that changing the pH value can be irritating to human eyes and might even be corrosive, leading to serious damages such as blindness. It is recommended to keep the product’s pH at a neutral level. This is exceptionally important.