Team:NEU CHINA/Human Practices

HP_Integrated Human Practice

HP
Integrated Human Practice
OVERVIEW
Between April and May: Survey
June: Industry
    Therefore, besides on constructing an engineered bacterium itself, it is also important to put our bacteria into practical usage in the future. The communication and the suggestion significantly enriched our initial ideas, as:
   1. Our “kill-switch” part is vital for biosafety problem, with the function of preventing modified genes from leaking into the natural environment. However, there is a severe leakage problem in “kill switch” that we need to improve.
     2. To sense the inflammatory signal, we can construct more sensors so that we can select one with the highest sensitivity.
     All the ideas above allowed us to purposefully design and model our system.

August: Publicity& Propaganda
Lecture at university
    As a therapeutic project we even want to characterize whether it will work on a real system, and we are very honored to have a professor , who is an Associate Professor at the University of Texas Health Science Center at Houston. She generously shared her knowledge of the gut microbiome and gave several suggestions on our iGEM project (Fig. 17).
We introduced our project to her after class and she encouraged us on continuing a further study. What’s more, she gave several advices for our future research directions (Fig. 18). She suggested that we should have a detection of whether the E. coli Nissle could colonize in the gut of mice and how would it behave in a healthy vs. dysfunctional gut microbiome? In addition, she taught us so many knowledges about gut microbiome.
September:Visitation & Caring
April: Hospitals
Graph 2. The pie graph of the acceptance of virous treatments.
Figure 4. Photos of Shenyang Lorean Biotechnic Company .
July: Professor Interviews
Figure 5. Talk with Pro. Fei.
Figure 6. Talk with Pro. Yu.
Figure 7. Lecture about synthetic biology and iGEM given to the freshmen in NEU.
Figure 8. Heated discussion with the students.
Social Media
Figure 9. The photo of an interview on the local television.
    Under our vigorous propaganda, our project has attracted a wide attention from all walks of life in Shenyang. To this end, Shenyang TV invited our teammates and our mentor Prof. Chen Ding to participate an interview program which called “Our Community Our Home” (Fig. 9). We were glad to see what we were doing had been recognized by people. By the way, the interview itself was also a good medium for exporting our projects. And we hope more and more people will have a deeper understanding of IBD and pay attention to it after they know what we are doing.
HTML 5
    We also designed HTML 5, a kind of web page on the Internet, and spread them on Wechat, QQ, Instagram and some other apps and websites, in order that more people can get familiar with our project as well as probiotic therapy. People showed great interest in our pages and left us numerous messages about the knowledge that they wanted to learn more about probiotic therapy. We gave them replies and some details under each comment.
please click here(pdf.) to see our HTML 5.

Global Grand Challenges Summit 2019
Background:
    Global Grand Challenges Summit 2019 is held in London, United Kingdom. Every year, engineers, politicians and entrepreneurs from China, United Kingdom and United States of America will attend this summit to evaluate the possibilities of bio-engineering medicine in the pharmaceutical market.
Topics were included in this year:
1. Will Artificial Intelligence (AI) and other transformational technologies change humanity for the better life?
2. Can we sustain 10 billion people in the future?
Student collaboration:
   One of our team members, Wu Shuang, was nominated to attend this big event. He participated in the international student team named “greenGut” , which consisted of 5 students from China, the US and the UK. The team mentor was from the University of Edinburgh. Focusing on providing better health care and engineering better medicines, Wu proposed a new diagnostic method for IBD based on his current work for his 2019 iGEM team NEU-CHINA. This new product, named greenGut, is an bio-engineered probiotic strain. Since the concentration of nitric oxide in IBD patient’s gut can be 100 times higher than that in normal people, Wu introduced the nitric oxide inducible promoter PyeaR and a GFP gene as the reporter gene. In that case, after the strain successfully colonized in the colon, the IBD induced nitric oxide secretion could trigger the PyeaR promoter activation, expressed GFP reporter gene, which would indicate the IBD status. Wu and his team greenGut won the first place in student collaboration and finally made it to give a pitch presentation in front of engineers, politicians and entrepreneurs from China, British and United States of America (Fig. 10, Fig. 11). During this event, he spoke with mentors, professors from Royal Academy of Engineering (UK), Chinese Academy of Engineering (China) and National Academy of Engineering (US) and got precious feedback to improve his strain, greenGut and our iGEM program (Fig. 12, Fig. 13).

Figure 10. Twitter from Royal Academy of Engineering reporting final presentation of team greenGut.
Figure 11. Team greenGut. The first person on the right is their mentor Ignacio Tudela-Montes Dr. from University of Edinburgh.
Figure 12 and 13. Final pitch presentation at the main hall of GGCS in front of engineers, politicians and entrepreneurs from China, British and United States of America.
Figure 14. Talk with mentor from Royal Academy of Engineering.
Feedback:
    As the mentor of Wu’s team, Dr. Ignacio put emphasis on the cost of our engineered probiotic drink. “The most tough challenge for IBD is that, current diagnostics are expensive, painful and can sometimes misdiagnose.” he said, “Therefore, you have to make sure that your new product for IBD diagnostic must be cheap and accessible enough to win a place in the huge market.’” When talking about Wu’s iGEM team, who were aiming at a better way to help ameliorate IBD, he said that we were facing the same question: did we really make it much cheaper to treat IBD? He suggested that we contacting with the National Medical Products Administration to see the cost of commercialization of our product (Fig. 15).
    During the communication with a professor, he emphasized on the sensitivity of the nitric oxide sensor, as it should be sensitive enough to detect in concentration of nitric oxide. “You don’t want your probiotic to express protein at wrong timing, right?” he said. In addition, the cost also needed to be put into consideration, which means that it is somehow necessary for us to learn how to commercialize our product from pharmaceutical factory (Fig. 16).

Figure 15. Photo with professor, fellowship of Royal Academy of Engineering.
Figure 16. Photo with the Professor Xv Sheng from University of California, San Diego.
August: Publicity& Propaganda
Figure 17. Photo with Dr. Krachler who gave us the lectures about gut microflora.
Figure 18. Talk with Dr. Krachler.
    We paid a visit to the community nearby, where we sent the organic broccoli that planted on our own, hoping to arouse attention of forming a healthy eating habit (Fig. 19).
Figure 19. The visit to the community nearby.
    Through literature review, we learned that an adequate intake of cruciferous plants such as broccoli can effectively prevent intestinal disease and alleviate inflammation. Therefore, we established an ecological garden to plant various organic vegetables, including broccoli, tomatoes, and cucumbers and so on (Fig. 20).











Figure 20. The photo of broccoli harvest.
     In July, we started planting broccoli with the help of gardeners from our college. After nearly 60 days hard work, broccoli was ripped in September and we harvested approximate 100 kilograms in the fields. We brought our organic broccoli to the community and introduced the healthy diet; meanwhile, we introduced the benefits of broccoli in gut inflammation prevention.
   After visiting to the local residents, we knew that most of people, especially the aged, were willing to strengthen their body by consuming healthy food. However, some of them were optimistic about the treatment of gastrointestinal diseases. By conducting this activity, we hope to call on people around us to lead a healthier life.



    In October, the last period of our project, we paid a visit to the Drug Administration to get the knowledge of the drug management system, so that the drug we designed in our project can be produced and utilized clinically in the future.

    We have been expecting that our project products can be truly applied to clinical treatment and developing the drugs. In order to understand the government's regulations on the market entrance of biologicals as well as their current market and application forms, we visited the local drug administration to interview the staffs and collected more information.
      The staff told us that most of the biological products that currently used on the market for clinical treatment are monoclonal antibodies produced by engineered bacteria and other drugs. We noted that no live probiotics in biological products have been used so far for clinical treatment. In addition, he also mentioned that there were only about hundreds of biological preparations approved for market at home and abroad, while a lot of biological preparations are being studied. Compared with traditional chemical synthetic drugs, the number is obviously very small.
     What’s more, the staff also stated that the development and sale of any drug, including synthetic drugs, is a long process. They made several comparisons between chemical synthetic drugs and domestic biological products. Chemical synthetic drugs, due to its short R&D time and mature production technology, developed further faster. By contrast, source shortage and complicated production process, limited the pace of the biological’s development. As for the conditions for listing, there is no significant differences between domestic biological products and chemical synthetic drugs. Compared to the chemical synthetic drugs, only bacteria, viruses, cells and some other biological raw materials are extra required to get approved to listing, while the rest is the same as the chemical synthetic drugs. As a result, biological products have a very broad prospect for development.
    We introduced our project to the staff of the drug administration, using live probiotics to treat diseases, a new concept of medical treatment, and has been affirmed by them. They emphasized on the significance of biosafety problem in drug research and development. In the production process, including when drugs enter human bodies, every aspect of safety should be taken into account. Moreover, because of their instability, the storing of biological agents should also be concerned. In the future, the methods of maintaining its production, transportation, storage and stability inside human body may also be the focus of research.


Consulting Pro. Yu
Consulting Pro. Sheng
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NEU-CHINA
    With the accelerating pace of modern life, more and more people have developed bad eating habits, which lead to a growing emphasis on the gastrointestinal issues. Among these gastrointestinal issues, the inflammatory bowel disease (IBD), a chronic inflammatory condition of the gastrointestinal tract, is one of the serious health concerns today. Up to now, there have been five main categories of medications used to treat IBD, which are aminosalicylates, corticosteroids, immunomodulators, antibiotics and biological therapies. However, current therapies are unable to relieve established intestinal fibrosis. Because the intestinal microflora acts as the key role in the development of intestinal inflammation, traditional drugs are either targeted at the host cells or to emilite the microflora around the inflammation sites to relieve the symptoms of IBD, both of which ignore the intestinal microflora’s effects on the inflammatory bowel disease. Therefore, we think that impacting the microflora may be a possible therapeutic approach. Then, we come up with the idea to utilize the engineered bacteria as a novel therapy. To arouse more attention to this therapy as well as get some suggestions to our project from other people in kinds of areas, we visited local hospitals, delivered several public speeches and established an ecological garden. What’s more, we also cooperated with NEU Rubik’s Cube Association, set up Talent Project and lab workshop, designed comics and video games as fancy education programs.
    Here is the map that summarize our human practices:

Figure 1. The human practices map of Team NEU_CHINA
    From April to October, we have been dedicated to improving and propagandizing our project by getting more people involved in our project and collecting a wide range of suggestions. By doing this, we kept persisting a more perfect project that can meet more people’s needs and make a difference to the world.
    In order to understand IBD more clearly, we went to Deji Hospital in Shenyang to exchange our project with Dr Wang, an expert in internal medicine (Fig. 2). We asked her several questions about the current clinic methods and the difficulties for IBD treatments.
    From the interview, we learned that treatment medication is the first therapeutic option. So far, five categories of medications have been used in the therapy of IBD, which are aminosalicylates, corticosteroids, immunomodulators, antibiotics and biological therapies. However, there is no standard IBD therapeutics and there are several challenges of this disease.
    On one hand, as the chronic and life-long disease, IBD has been demonstrated with a great risk association with other diseases, including colon cancer, blood clots and a liver disease called Primary Sclerosing Cholangistis (PCS). On the other hand, the mental and financial burden is another obstacle for the IBD therapy. Once the patient is diagnosed with IBD, not only should them shoulder the high costs of the treatment, but also suffer symptoms of painful and uncomfortable, which is likely to lead to the elevation of stress level. Therefore, it is still a long way to go to cure the disease.
    After the interview, we were even more determined to make efforts for the prevention and treatment of IBD. But as a therapeutics program, how to release the drug, where to release it, and what's the public concerns are what we're going to explore in the next step.

    We conducted several surveys between April and May, in order to collect more suggestions and considerations for our design and the propaganda afterwards. We hope that by receiving all the replies, we can have an understanding of public needs for our product so that we can focus on the specific aspect more on our project.
  

  Last year, we casted questionnaires on several network platforms and the feedback told us that most people had a limited understanding on the gastrointestinal disease. This year we continued to dedicate to popularize the knowledge of IBD as well as some other gastrointestinal problems, with an increasing perception to the IBD from the public. As a result, as for the survey we launched this year, we included some more concrete issues.
    At the early stage of our project, we issued hundreds of questionnaires on the internet. The questionnaire investigated on the question, “What’s the first three diseases you can think about when having a gastrointestinal uncomfortable?” with several common intestinal disease listed on it. From 198 replies, we plotted the outcomes in graph (Graph 1.).
Graph 1. The answer distribution of question “What are the first three disease you can come up with when having a gastrointestinal uncomfortable?”
    The result demonstrated that diarrhea, indigestion and gastritis were the top three sufferings that came into most people’s mind. What’s more, we were delighted to find that more people can think of IBD than last year beyond our expectation, which proved an effective publicity and increasing concerns on IBD. With a broader knowledge of the intestinal disease, people’s life can become healthier.
   The treatment of IBD has become a medical hot topic in western countries for many years. However, statistical recorded on the ACCESS, which is issued on ‘Gastroenterology’, indicates that the morbidity of IBD in China also tends to increase, requiring more social attention. Therefore, we also conducted a survey on suggestions and inclinations for IBD drugs among Chinese population.
    In the thousand years of development on medical treatment around world, we can recognize an obvious difference between western and traditional Chinese medical on the preference for medical treatment. The acceptance for surgery and injection is generally lower in Chinese medicine than that of western due to couple of reasons. On the one hand, the centuries-old history of oriental medicine deeply affects people’s acceptance and cognition to some foreign medical treatment. On the other hand, some traditional views and customs also prompt a higher favor for oral medicine than injection or surgery (Graph 2.).
    Among the IBD medical treatments, apart from utilizing drug therapy, such as aminocalicylic acid, corticosteroids, immunoregulator and antibiotics, we realized that surgery became necessary to patients after a long-term suffering, which may lead to tension both physically and psychologically. As a result, a new type of drug needs to be developed to alleviate the inflammation symptomes.

   Among the clinical proved drugs, not all of them can have specific therapies to IBD. Besides, as to the several traditional chemosynthetic medicines, we recognized that people were felt an expression of greater interest and expectation to biological therapy, which own infinite potential and development in the future. They asked several questions while we explained our initial idea of utilizing engineered bacteria for IBD treatment. In order to have a more impeccable understanding of the focus that people concerned about biologicals, we casted questionnaire not only on 50 patients, but also 30 biopharmaceutical manufactures, hoped to know about various perspectives from two different angles (Graph 3.).
Graph 3. The most important factors of a new therapy of IBD from two group interviewees, patients and developers.
    The results showed an increasing acceptance for biological therapies, which made it a bright future for novel medicine development. With the rapid development of medical approaches and cognition to diseases, many drugs for treating gastrointestinal disease could be created.
    Based on the project of NEU_CHINA_A 2018, we intended to develop an engineered bacteria-based therapy that enables to alleviate the gastrointestinal inflammation. We visited the Department of Gastroenterology in Deji Hospital in Shenyang, where we conducted a survey for evaluating the possibilities of our bio-engineered therapy from doctors and IBD patients.
    There are various types of drugs for intestinal diseases. From questionnaires, we found that combining the drug with drinks was the most acceptable administration route (Graph 5.). Next, we also launched the favorite flavor of the “drug drinks”. It showed that vanilla flavor was the most popular. Some other flavors, for example, fruits or cocoa rank second to it. These details might help us steering the direction of developing our future product so that we could modified the future product according to the findings (Graph 6.). 

    Biological products occupy large pharmaceutical market, with an increasing of 70% sales in the past years. With its rapid development, it also has immense potential in therapeutic field, efficacy and practicability, which shows that it has a broad prospect. At present, many global pharmaceutical companies are doing researches on drugs in this field. With a more mature therapy developed by biotechnology in the medical field, the developing potential of biological industry can be better manifest.
Graph 5. The most accepted forms of intestinal drugs.
Graph 6. The favorite flavor of drinks.
    By visiting the biotech industry, we recognized the significance of the practical application value on our synthetic biology project, which gave us several inspirations to construct our engineered bacteria.
    We went to Shenyang Lorean Biotechnic Company on Jun 10 (Fig. 4). By chatting with the product research people, we learned that probiotics with various target functions have become popular with deepening of research and the increasing of acceptance. However, no modified probiotics have been approved yet because of the high risks and the immature technology. Only some biosafety issues can be solved by the engineered bacteria in the market.
    Having a immature frame of our initial design, we asked for more advice from the professors to get some improvements of our design.



    Next, Professor. Yu, an expert in protein expression using microorganism, we asked her for help to demonstrate western blotting (Fig. 6). In our early experiment we were having trouble to see a result after chemiluminescence, the protein seemed to be so sticky that it stuck in the top of the gel instead going down along the lane during electrophoresis. She then made a conjecture that it can be due to the high concentration of protein sample. Also, destruction of cells by sonic oscillator should be removed from the protocol, since we are running protein gel using whole protein sample, lysis of cells makes no difference if we load the whole cells directly after incubating at 98℃ for 10 min. She said that, if we span down the protein sample after ultrasonic crushing at 13000rpm, insoluble protein will become sediment along with organellas, which means that if you load the supernatant to the protein gel, you are lossing unsoluble protein. What makes things worse is that, expressing exogenetic protein in bacteria is difficulty and can result in becoming insoluble protein which means no bioactivity. Suggestion given by her is to incubate cells culture at a low temperature around 16℃, this will slow down the expression process to give more time for protein to fold to reach the correct conformation. Bioactivity is supper important for we are expecting the medical use of protein.
    We interviewed Pro.Sheng, discussing the leakage problem of our NO sensor. Above all, he recommended that more types of NO sensors can be tried so that we could select the one with the highest sensitive and the lowest leakage. In addition, he agreed that adding another NsrR Binding sequence as a ‘Road Blocking’ effect was a good choice for leakage of nitric oxide sensors. What’s more, he also pointed out that the distance between the extra binding sequence might have some influence on the final result. What he said prompted us to undertake further research into the NO sensor in our project.

    To strengthen publicity and acquaint them with IBD as well as synthetic biology, we delivered public speeches at schools and via social media, which had attracted numerous people.


   We delivered an interactive presentation at our university on September 7th, aiming to spread knowledge about IBD, some basic concepts of synthetic biology and its development prospect (Fig. 7).
    A large number of students were attracted by our topic and conducted a heated discussion after the lecture (Fig. 8).
    It is recognized by the participants that this application has a very wide range of prospects and commercial value.

Figure 2. Talk with Dr. Wang.
Figure 3. Talk with IBD patients.
Graph 4. The attitudes of public on engineered products.
    When it comes to biological therapies, the statistic shows that patients pay more attention to safety, price and efficacy; while pharmaceutical companies take more consideration on safety and profits. However, safety is the very important problem for both patients and manufacturers. Among the genetic products, from food to drugs, must be undergone a series of rigorous examinations and security assurance before marketing. We also intended to emphasis on minimize biosafety problems of our project in light of all the results above.
Before that, we needed to figure out the public attitudes about genetically engineered products in advanced, so we issued the questionnaire to the residents within various social medias (Graph 4.).

Consulting Pro. Fei
    We interviewed Pro. Fei, who is an expert in gene editing (Fig. 5). He highlighted the importance of controlling the concentration of drug delivery. He pointed out that a part could be constructed as a rotary switch, so that the expression of our drug can be precisely adjusted. He provided us with some readings of the ways to control drug delivery, which may inspire us to design our new part. What’s more, he emphasized on the biosafety problem which can be focus in the public. His suggestions and guidance were pivotal to our construction to our Tuner part.