Team:QHFZ-China/Human Practices
_HUMAN PRACTICES_
Summary
Overview
After communicating with our relatives, we found that some of them suffer from symptoms of gout. To better understand the condition of gout in the world, we searched on the internet to read news, reports, literature and books. We have learned that thousands of people are experiencing the effects of gout, and more people have hyperuricemia. The common cause of two diseases is the accumulation of excessive uric acid (UA) in the body. In order to reduce the pain of these loved ones, we decided to do something to help them prevent or treat gout: that is, to build an accurate and sensitive detector for uric acid detection, and an efficient smart cell for uric acid degradation.
To confirm the current situation of gout and the significance of our project, we interviewed Dr. Liu and Dr. Wang. We learned about the drawbacks of the current methods of UA detection and gout treatment. For UA detection, the blood test in hospital is always time consuming and expensive, and the process needs complex instruments and professional operators, which limited the application of blood tests.
Meanwhile, the common method to treat hyperuricemia and gout is using drugs. However, drugs are expensive, and need to be taken for a long time, which is easy to cause damage to other organs and tissues, and it is also difficult for patients to take medicine regularly. These results make us understand the importance of the project and motivate us to complete the project design.
In the process of designing, we communicated with two past iGEM teams, Hong_Kong_UCCKE 2017 and DLUT 2018, which studied gout in their projects, to get their experience. Their suggestions helped us to avoid mistakes and select useful bioparts. (We record this work in Collaborations section)
After sharing the preliminary data with an expert majored in biosensor, we found our design still has the following problems, including long waiting time and low sensitivity for clinical usage. It promoted us to optimize the UA detector by introducing an amplifier. To improve our UA degradation design, we interviewed a scientist majored in stem cell therapy, to get methods which may help the product meet the requirements raised by the doctors and the public. Their advice provided us guidance for our future plan, such as introducing a switchable biosafety control system, encapsulating the engineered cells if we hope to use the design in vivo, etc.
Considering that the stakeholders of the project mainly contained doctors, patients and the public, whose needs and requirements are very important for us, we designed a questionnaire to get public advice and opinions on our project. 460 feedback provided us with valuable comments and suggestions that could guide our future plans.
Human practices have promoted our projects from beginning to end. In addition to the main line of human practices, we also did some other works, as shown in the following table.
Integrated human practices
Part 1: Disease research
1. Interview with experts who major in hyperuricemia and gout:
Interviewees:
A. Wei Wang (A doctor in kidney internal medicine department in Changgeng Hosipital in Beijing, China.)
B. Ruixue Zhang (A doctor in gout outpatient in Huaxin hospital in Beijing, China.)
C. Mr. Liu (A doctor in rheumatology and hematology department in a hospital in Fujian, China.)
Form: Offline interview (Dr. Wang and Dr. Zhang) and online communication (Dr. Liu).
Summary:
We asked the current situation of hyperuricemia and gout. The doctors told us many useful information: (1) Because of the changing lifestyle, the number of patients suffered from hyperuricemia and gout increases gradually. (2) The current medicines have more or less toxicity to liver or kidney. Some medicines, such as allopurinol, are high-risk allergen to Asian. Some medicines, such as colchicine, only give patients temporary control. What’s more, many patients used irregular medications, which limits the drug efficiency. (3) To test patients’ serum uric acid (UA) concentration, people need to go to the hospital at morning and get the results at afternoon. The cost is 20 to 30 RMB. Long time waits and high medical expenses make people expect a better alternative method to detect the UA concentration. (4) Hyperuricemia would harm the body more than gout, such as causing angiocardiopathy, chronic nephrosis and hypertension. It is necessary to control the UA level in body fluids.
We also asked their opinions to our project. Above all, they thought the project was meaningful and could help people to beat hyperuricemia. However, they suggested us to do more improvements: (1) Cells should stay as long as possible in the body, in order to reduce frequency of cell injection. (2) The system needed to be optimized to reduce the negative effects of injection. (3) If we want to use bacteria as a reporter to detect UA concentration, the genetic circuit should give out fluorescence signal as quickly as possible, in order to reduce the waiting time of the users.
What we learned:
(1) Our project is deserved to carry out to enrich the means to fight hyperuricemia and gout.
(2) We should enhance the retention capacity of the engineered cells. By literature review, we found it just need to change only one single base of the NRF2 gene [1] which could improve the retention ability. We will try it in the future.
(3) It is necessary to interview scientists to learn about the methods to avoid rejection and how to make the UA detector response faster.
2. Visiting a biotechnology company
Interviewee:
Yuxiang Yan (CTO of Pysma Healthineer, which aims at making a non-Invasive uric acid monitoring and alarm system)
Form: Offline interview.
Summary:
Dr. Yan firstly shared his investigation about gout. The current situation of the disease is serious. Up to 4%-6% of the population suffers from gout, especially in some coastal cities of China, such as Dalian and Qingdao. What’s worse, most people know little about gout, so that the diagnosis and treatment were delayed. They don’t eat medicines. They even don’t know which department to go to visit a doctor.
We interviewed Dr. Yan to know the mechanism of their commercial instrument and how to achieve non-invasive. He answered that the key component they used is uricase, which has been widely used in the hospitals and uric acid detection kits. And the sample they test is saliva, instead of blood, which is non-invasive.
What we learned:
(1) Our project is meaningful. On the one hand, the current situation of the disease is serious. On the other hand, there are some companies and people studying in this field, which verifies that our idea is not ridiculous.
(2) We need to come out an effective method to publicity the knowledge about hyperuricemia. For this purpose, we need to interview a publication editor.
(3) We realized that it would take a long-time scientific research to clinical application. Though uricase has been used wildly, Dr. Yan still needs to do much work to build a better machine. For us, though we designed all parts of the product, we should realize that our there are still many works to optimize before application.
(4) Through this visit, we learned that in addition to measuring blood samples, there are other types of samples available.
3. Questionnaire survey
Interviewee:
Juan Du (A doctor in Department of Psychology of Tsinghua University, who is an expert in questionnaire design and analysis)
Summary:
In order to understand the public's views on gout, uric acid detection and cell therapy, we designed a questionnaire and spread it. We got 460 answers in total. The answers came from 29 provincial-level regions of China, as well as 17 answers from foreign regions (however, as we only spread the questionnaire by Wechat, which is a communication software only used in China, the 17 answers could be generated by some mistakes.); the male versus female is 1:1.3; the age distribution is: 21% below 22, 46% 23~45, 32% above 46; at last, 19% are gout / hyperuricemia patients (which is close to the morbidity in China, which indicated that the sample could represent the totality). Though the distribution of the answers was not absolutely ideal, we considered the result made sense, as the answers came from all kinds of people at an acceptable proportion.
To evaluate the validity of the questionnaire, we interviewed an expert at questionnaire. She told us our questionnaire was significant in general, though some details were not enough professional. Then she shared knowledge about questionnaire design and analysis. In addition, we also confirmed the rationality of the questionnaire during doctor interview. By these helps, we analyzed the result of our questionnaire.
(We tried our best to protect the privacy. We avoided surveying personal information that are not suitable to ask in China. What's more, we invited some persons to check the questionnaire before we spread it and people were voluntary to decide whether to answer it. However, during the Jamboree, the judges told us that in the future, we should pay more attention to protect the privacy. In China, the Irb is not well-known. However, in the future, before spreading a questionnaire, we must understand Irb further. Everyone has the responsibility to propagandize Irb in China to do a more reasonable work.)
What we learned:
(1) Only 17% people resisted to use the intelligent cells to help control the uric acid concentration if their UA concentration was abnormal, which implied the acceptability of cell therapy was even higher than our expectation, which indicated our project would meet less resistance and get more value.
(2) 44% of people above 46 years old didn’t know stem cell technology, at the same time 27% of them resisted cell therapy. On the contrary, only 23% of people below 46 years old didn’t know stem cell technology, and only 13% of them resisted cell therapy. Obviously, more and more people in China, especially the young people at school, would know more about biology and accept new medical methods. To us, we would pay more attention in popularization of science. For this purpose, it is needed to interview a publication editor.
(3) 74% people didn’t like to go to hospital frequently to treat the disease, which again meant that we should make the intelligent cells to stay in the body longer.
(4) 78% people didn’t like to go to hospital to detect uric acid concentration more frequently than once per month. 83% people wanted to reduce the cost of the detection to 10 RMB once. However, as we knew from the doctors, the cost was 20 to 30 RMB. So, our project would give people a better choice.
(5) Only 12% people didn’t think adding a suicide switch in the cells could ensure safety. As a result, we will add a suicide switch in the future to meet most people’s viewpoint. However, we will also do more to satisfy all people. We subsequently interviewed a scientist to learn how to achieve it.
4. Information collection about gout and the patients
Activities:
To learn more about the disease, we searched on the internet, read books and literature, and communicated with doctors and our parents. To learn the feelings of the patients, we indirectly got the information from our parents, the doctors, the internet and our questionnaire.
Summary:
We read a lot to learn the mechanism of gout and related medicines. Gout was very, very, very painful. However, hyperuricemia and gout are hard to cure. The patients even don’t know if they will pain today and they are always worry to gain renal failure, which extremely influences their psychological health. Patients are tired of eating pills every day, so they want us to develop a method to control uric acid level for a longer time than medicines. Meanwhile, the diet was highly limited to the patients, which they feel is very hard to insist. In addition, there appears a kind of portable uric acid detection device. However, by using it, they found it is expensive but not accurate enough. As a result, it is necessary to develop a new way to monitor the uric acid level.
What we learned:
(1) Though it is said that the disease is highly under control today, to patients, the existing methods are far from beautifying their life. If our project works well, it might give them a light.
(2) It is necessary to optimize the gene circuit to make it more accurate and cheaper.
(3) From the questionnaire, some participants left messages that they were looking forward to new treatment methods. That gave us a lot of power to accelerate our study. However, we are still senior high school students, and we clearly know that what we are doing was still far away from application. So, we decided to do more both during and after the iGEM tournament, and share all information we got from our interviewee.
Part 2: Project optimization
1. Stem cell scientist interview
Interviewee:
Haoyi Wang (click his photo to get his introduction)
Form: Offline interview.
Summary:
We asked ethical issue of stem cell therapy. Dr. Wang said that using the technology might be OK in the field of medical treatment, while using it in reproduction system or embryo may touch on ethical issue in China.
We described our design to make a smart cell to degrade uric acid. Dr. Wang approved the design. However, he gave two pieces of advice. Firstly, instead of injecting the cells into blood and joints, if we could make a micro organ, the safety could be better ensured. Secondly, he hoped us to estimate the cells we need to inject into the patient’s body and confirm that the quantity was acceptable.
We asked if it is possible to use iPS cells from the patients to make the intelligent uric acid-degradation cells, so that the rejection reaction could be avoided. Wang told us that we did not need to use so much effort, because in the future, people would have stem cells with all kinds of matches. We can use the cells to develop our designed cells. To the patients, they only need to test if they matches and use cells with suitable matches.
What we learned:
(1) After the interview, we decided to use smUOX to replace UOX, as smUOX can be secreted out of the cells to degrade uric acid. In the future, we will design a micro organ and package cells in biofriendly materials, only smUOX can get out to the blood and joints to degrade uric acids and uratoma.
(2) We will try to enhance the UA degradtion efficiency and then estimate the needed quantity of smart cells by modeling with the data, as well as referring to the data from related literature [2].
2. Plasmid request for circuit optimization
Interviewee:
Shuangyan Tang (click her photo to get her introduction)
Form: Communication by e-mail.
Summary:
By literature search, we found a suitable gene circuit to detect uric acid [3]. To get the permission to use the plasmid, we sent an e-mail to Dr. Tang, who was the corresponding author of the article. Dr. Tang selflessly permitted us to use the plasmid. Notably, the plasmid had an intellectual property.
3. Biosensor expert interview
Interviewee:
Jack (Xiaoyu) Chen (click his photo to get his introduction)
Form: Online communication, together with BEAS_China 2019.
Summary:
We described our design to make a bacterium to detect uric acid concentration in blood and saliva samples. Dr. Chen told us that serum and other substances in the samples were likely to influence the response of the biosensor or the vitality of the bacteria. As a result, the samples should be highly diluted by broth. So, the biosensor must be able to detect uric acid at a very low concentration. Therefore, an amplifier module should be added to enhance the sensitivity. However, though multilayer amplifiers could obviously enhance the sensitivity, it is hard to debug. As a result, we should start at one-layer amplifier.
We asked another question: how to reduce the response time of the biosensor. Dr. Chen gave some useful advice. First, we can try to change a fluorescent protein with a shorter maturation time. Secondly, we should look for a suitable condition, including adjusting the components of the broth, such as the bacterial density, etc.
What we learned:
(1) To enhance the sensitivity, we could add an amplifier, RinA_p80α – PrinA_80α system to the original gene circuit [4].
(2) To reduce the waiting time of the users, we changed the dsRed protein in the original gene circuit into superfolder GFP (sfGFP), which has a quicker folding speed.
(3) In the future plan, we will do more condition optimization to accelerate the response.
4. Engineering professor interview
Interviewee:
Weiqun Yang (click his photo to get his introduction)
Form: Offline interview.
Summary:
We wanted to get some advice to build our hardware, so that the users can read the fluorescence of the biosensor directly. Dr. Yang firstly warned us that hardware building was a very difficult work. After he listened to our description about project, he advised us to pay more attention to the bio experiments, because a stable and reliable gene circuit is the foundation of the uric acid detector. To spend less time on hardware, he advised us to take advantage of the products in sale, instead of building it start from scratch. In this way, we could build a simple fluorescence reader to deal with the information sent out by our bacteria.
What we learned:
(1) We adjusted our plan on hardware. Instead of making a product, we will focus on investigation and preliminary test for proof of concept.
(2) We would use the sensor and processing system of LEGO to build our hardware, instead of building photoelectric circuit ourselves.
Public Education
Most work we did in the section was done during Tsinghua university student carnival, during which a lot of junior / senior high school students and teachers visited our laboratory and we showed several prepared programs to them to spread the knowledge about synthetic biology.
(1) Let public try to extract PHA from E. coli cells.
Polyhydroxyalkanoate (PHA) is a substitute of biological plastic, which can be produced by bacteria and is degradable. To promote environmental awareness, spread the knowledge about synthetic biology, and let public experience experiments, we borrowed the strain from Bluepha Company and designed a rapid PHA extraction experiment.
During the experience, we didn't stop to tell the participants about laboratory safety operation specification, the use of related equipment, and knowledge about biodegradable plastics. (We apologize for the chinglish sentence that confused the judges in the Jamboree. We meant that we keep emphasizing the safety and related knowledge to the participants.) The participants extracted PHA from the bacteria by dishwashing liquid, and we helped them package PHA into a postcard as a souvenir.
(2) Let participants experience bacterial paintings
Painting is very attractive to everyone. As a result, we took advantage of bacterial painting to interest them in biology and spread related knowledge during painting.
Before painting, we used the flashlight for some finished products with superior bacteria daub of medium to illuminate, and on the principle of bacteria can shine, many young people felt incredible after listening, and showed a strong curiosity, and this curiosity also performed on projects we do it. We collected their work, took it into the bacteriological incubator in our lab for overnight cultivation, and returned the finished photos to them. The strains were provided by Bluepha Company.
(During the Jamboree, the judges worried that the participants could take the bacteria away from our lab. We declared that it did not happen. The participants must wear gloves and we were keeping monitoring them during the experiments. To bacterial painting, their painting were collected by us and we sent them a photo. To PHA extraction, the bacteria were killed by lysis buffer and high temperature. And the extracted PHA was sealed in a postcard by a plastic-envelop machine. However, in the next year, we will give a better design to make the participants much further away from the bacteria to keep their safety.)
(3) Show the Lego fluid shifter we made -- describing the principle and use method
Pipette is an important tool in experiments. At the same time, its principle is not complex. We planned to introduce pipette to the public. To make the process more attractive, we made a pipette with Lego, which can freely adjust the amount of liquid absorbed, and can easily remove the used gun head. Due to the exposed structure, it is also helpful for visitors to tell the principle more vividly.
(4) Publicize synthetic biology to visitors
We set up a display board to introduce synthetic biology, iGEM and out project to the visitors. The visitors said that they were interested in it and would get more information about synbio and iGEM on the internet. Moreover, we have collected a lot of their perception of synthetic biology. Many of their proposals and suggestions for our project has played a big help. For this section, we made many kinds of delicate fridge magnets as souvenirs, on which we printed biology related pictures (DNA, PCR amplifier …). After our introduction, they became to pay attention in this field.
(5) Popularize gout related knowledge on the internet
In order to improve people's understanding and prevention of gout, we sorted out the knowledge we collected and the information we obtained during the interviews, and put it on the website of Zhihu, hoping to attract people's attention through this media communication mode and improve some bad habits, such as sitting for a long time and drinking. On another website, bilibili, which is a video website, we posted some videos about our experimental process and team building activities, so that the general public can learn about synthetic biology, bioexperiments and our team.
Other human practices work:
Summary:
(1) We attended the after iGEM Meetup 2019 held in Beijing. We introduced our project to many iGEM 2019 teams and they gave us a lot of useful information.
(2) Our instructor, Cheng Li, gave us a lecture once a week during February to June, to make us understand synthetic biology and know how to design a project, do experiments and analyze data.
(3) We interviewed a senior editor, Yizhu Wang, to ask how to do art design and how to make people know more about the disease. Wang told us that new media and media matrix should be utilized, together with careful and creative design. However, there is no difference between simple science popularization and no science popularization.
(4) We visited Bluepha, the most famous synthetic biology corporation in China, to learn the current situation of biotechnology companies, and how to promote industrialization of innovation achievements. Teng Li, CEO of bluepha, as well as our secondary PI, answered our questions and told us that we must confirm that our product is the best solution to this problem; if not, find the real value of our product.
(5) We invited two senior fellow apprentices of our high school to share experience to us. They are Yuwenbin Li, who is a Ph.D candidate in USCD and has attended iGEM twice; Ruiming Zhang, who has just got a gold medal in National School Biology League of China. They shared their experience in experiments and the iGEM competitions, which benefited and encouraged us a lot.
(6) We communicated with multiple iGEM teams in 2019, 2018 and 2017 and gained a lot of useful information, which will be detailed in the Collaborations section. As far as we know, we have communicated with all teams which used HucR and aimed at defeating gout. (We should apologize that we did not find that Hong_Kong_LFC_PC 2019 also devoted themselves to treat gout before the Jamboree. However, the two teams had a good talk and exchanged igeas during the Jamboree.)
(7) To inherit the spirit of iGEM, we set up an iGEM club in our school. For the next year, Team QHFZ-China will have a higher starting point based on our experience and efforts.
[1] Yang, J., Li, J., Suzuki, K., Liu, X., Wu, J., Zhang, W., ... & Qu, J. (2017). Genetic enhancement in cultured human adult stem cells conferred by a single nucleotide recoding. Cell research, 27(9), 1178.
[2] Kemmer, C., Gitzinger, M., Daoud-El Baba, M., Djonov, V., Stelling, J., & Fussenegger, M. (2010). Self-sufficient control of urate homeostasis in mice by a synthetic circuit. Nature biotechnology, 28(4), 355.
[3] Liang, C., Xiong, D., Zhang, Y., Mu, S., & Tang, S. Y. (2015). Development of a novel uric-acid-responsive regulatory system in Escherichia coli. Applied microbiology and biotechnology, 99(5), 2267-2275.
[4] Wan, X., Volpetti, F., Petrova, E., French, C., Maerkl, S. J., & Wang, B. (2019). Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals. Nature chemical biology, 15(5), 540.
