In order to ensure the feasibility, scientificity, practicability, logicality and value correctness of the project, in the four stages of the project (Selection, Construction, Evaluation and Application), we actively seek for different stakeholders and potential users in the upstream, middle and downstream. Ask for their opinions, help and guidance to conduct diversified and substantive integrated human practices.
Although most of our human practices can only be carried out online due to the restrictions of the epidemic situation, only a small part of our human practices have the opportunity to exchange and learn offline. We have still received many constructive feedback and key suggestions to optimize our projects.
On this page, we will introduce how to obtain suggestions in the structure of "overview - breakdown". And used to optimize the project, recording our struggle, inspiration, adjustment and efforts in the whole process of the project.
For sake of the purpose of this project is to build a series of gene originals that can achieve hypoxia concentration, the main purpose of our achievements is to help some experts and scholars in their research and development of synthetic biology enterprises, we have a lot of communication for them. In addition, we have also widely adopted suggestions from the public and netizens, and our partners have also helped us a lot.
"CLOSE THE LOOP"
Whether based on the value pursuit of our team or the high respect and attention to the results of the competition, we believe that a successful project needs to meet the interests and orientation of all stakeholders as much as possible. Therefore, our team fully referred to the suggestions of various stakeholders from the very beginning. In the project topic determination stage, after expert consultation, teacher guidance, literature review, public feedback, team communication and other processes, we comprehensively considered and excluded the infeasible ideas proposed in the "brainstorming". In addition, after combining the relevant principles of biological ethics, the project title was finally determined----oxygen hunter.
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Our team hopes to use synthetic biology to solve practical problems in life. Through observation and questionnaire survey of life, we found that hair loss has become a common problem for college students. Therefore, we hope to make a carrier material targeting hair follicles, so that drugs can play a role in human hair follicles. Out of respect and concern for the public's ideas, we made a questionnaire to investigate the public's attitude towards "using synthetic biology to treat hair loss" with the help of data analysis. However, the survey results are not satisfactory: the public still has many concerns and distrust about the use of synthetic biology to treat hair loss, and believes that there are also potential safety hazards in the clinical trials of this solution. The public's concern had a great impact on our idea. After discussion, we thought that the idea really lacked consideration of security and was not mature, so we gave up the idea.
Environmental protection related projects are also a key direction of our project selection. Inspired by the Great Barrier Reef protection project conducted by 2020 UNSW_ Australia team, we turned our attention to the ocean and proposed the idea of using biofilm to actively transport seawater for desalination. We have thought of two ways, one is diaphragm method, the other is absorption method. Due to the limited knowledge reserve, we consulted Bing Hu, the special associate researcher of the School of Chemistry and Chemical Engineering of Beijing Institute of Technology, and the secretary general of the Science Popularization Committee of the Chinese Society of Biological Engineering. But the teacher pointed out the problems of the idea sharply from the aspects of cell characteristics and desalination standards. Finally, with the teacher's advice, we gave up the idea.
At the beginning of the project appreciation, we focused on the project of the BIT China team last year and became interested in the taste that can give people a strong sense of happiness and satisfaction. So we proposed to biosynthesize 3-methylthiopropanol, which is mainly used for sauce flavoring, while improving the efficiency of its synthesis and reducing costs. In order to verify the feasibility and practicability of this idea, we had an online communication with COFCO through the introduction of the last BIT China team. But after listening to our ideas, COFCO experts denied our ideas in terms of application value, food safety, industrialization standards, etc. After much deliberation, we had no choice but to give up the idea.
In our daily study, we noticed that although nitrogen is one of the commonly used chemical materials, the most commonly used combined nitrogen fixation method is greatly affected by salt and alkali, and its survival rate is low. Therefore, we hope to use synthetic biology to make joint nitrogen fixation and salt tolerance, so as to promote the implementation of green and safe production of crops and the fertilizer efficiency of biological nitrogen fixation. However, through the communication with the team instructor Hu Liu and literature review, we found that we faced many and difficult problems on the issue of joint nitrogen fixation. With the existing knowledge level of team members, we may not be able to research available results within the time required by the competition, which made us have a preliminary intention to reject this idea. But we insist that biological nitrogen fixation plays an important role in agricultural production, and we hope to do our part, so we began to improve the idea of the project.
After the screening of the first round of "brainstorming" focused projects, we decided to conduct some questionnaires and opinions on the remaining projects to avoid the final project violating the value or criteria of biological ethics. In the questionnaire on the joint nitrogen fixation topic reserved in the first stage, we received more questions and concerns. Since we were planning to introduce the edited stress resistance gene into nitrogen fixing bacteria for application in soil, nearly 80% of the respondents expressed concern about this. They raised questions about soil safety, crop growth environment, human health and other aspects. Such investigation results make us pay special attention to them. After carefully reviewing the data, we found that the public's concern is not unreasonable. Although it is a pity, we still reject this idea.
After two considerations in the project topic determination stage, we have synthesized the opinions of all parties and the values and norms related to bioethics decided to combine the ideas of nitrogen fixation and hydrogen production. In view of the "oxygen paradox" and the requirement of synthesizing a tool for creating a hypoxic environment we decided the topic-- Oxygen Hunter.
The goal of our project is to use gene elements to build a local intracellular hypoxic environment, provide the required hypoxic environmental conditions for the intracellular reductive process or the role of specific enzymes, thus making it possible to use the "cell factory" to carry out nitrogen fixation, hydrogen production, carbon fixation and other production processes.
In the principle design of the experiment, we expect to build a complete "gene protein function" system to create a hypoxic environment on the premise of ensuring the normal metabolism of cells.
In order to monitor the intracellular oxygen concentration, we selected two promoters, nirB and ArcA, to build a "detection module" after extensive research. Based on the hypoxia induction mechanism of these two promoters, we use the former to monitor oxygen concentration in real time and the latter to monitor oxygen consumption rate.
We shared this idea with Professor Li Chun, who believed that the scheme was highly feasible and gave us full recognition. We also asked COFCO Group and Honghui Pharmaceutical about the design of the detection system, and they highly recognized our design and looked forward to our final results.
For the construction of "function modules", our idea is to use the leghemoglobin that can transport oxygen and the laccase that can consume oxygen (CueO) to build a hypoxic environment. We discussed this idea with Xin Fengjiao and other experts from the Chinese Academy of Agricultural Sciences, who analyzed our project from the aspects of safety, feasibility, and gave full affirmation.
In addition, we also communicated with Mr.Yang Yu, a special researcher at Beijing Institute of Technology, who suggested that we supplement the precursor substance γ -amino-butyric acid (ALA) for the synthesis of laccase and leghemoglobin.
To verify the effect of the detection module, we first need to create an external hypoxia environment. In terms of the design improvement of the experimental device, we consulted Yang Yu and Hao Ling , senior students of Beijing University of Technology, although the experimental device failed to be improved due to time.
Since most of the students in our team came from non biological majors, at the beginning, we encountered problems such as insufficient proficiency in experimental operation. For the PCR operation that is very commonly used in synthetic biology, we consulted GENEWIZ Enterprise. Members of the scientific research department of the enterprise answered our questions in detail and provided a lot of experience in synthetic biology experiment operation.
In all aspects of modeling work, we also had a lot of communication with all sectors of society. Because it seems (this is a fact) that experts and enthusiasts engaged in Internet work or research are quite active online but shy offline communication. Most of our communication with them is on the Internet. In addition, Xianwen Liu , a teacher from Beijing University of Science and Technology, provided advice on all aspects of our modeling process.
In the early learning of the use of the modeling tool MATLAB, the members of the modeling team learned the relevant open courses of the University of Science and Technology of China in Bilibili (known as "China Tubing"), which has yielded considerable benefits.
When modeling the oxygen diffusion model based on Fick's Law, we put forward relevant professional questions on Zhihu (the largest question and answer forum on the Chinese Internet) and got replies from some netizens.
When modeling the oxygen consumption of biochemical reactions, we need to solve the dynamic differential equations of the main oxygen consumption reactions in cells, and we encounter difficulties in the selection of solution functions. On the CSDN community (the largest programming discussion community on the Chinese Internet), we learned about the solutions of ode15 and sode45. Finally, after trying, we decided to use the ode15 solution.
When BP neural network was used to predict the oxygen consumption parameters of soybean hemoglobin, almost everyone in the modeling group had no relevant experience on how to build the BP neural network, so they turned to CSDN and Github again. With its help, after detailed literature search, Levenberg Marquardt (gradient descent algorithm) was selected as the training function and sigmoid as the activation function to complete the construction of BP neural network model.
After completing the basic tasks, we released the demonstration of the results of the modeling work and the explanation and science popularization of part of the modeling process on the Bilibili website, and received a considerable amount of praise and feedback. Some comments provided us with valuable suggestions for improvement, which benefited us greatly and helped to promote the subsequent improvement work.
After the experimental data were processed and analyzed, we first discussed with the instructors in the group meeting. After the preliminary arrangement, we also communicated with Mr.Yang Yu. Mr.Yang Yu agreed with and recognized most of the analysis of our experimental results, and also raised some questions and conjectures. After finding the literature, we completed the result analysis part of our project. Mr.Yang Yu is very optimistic about the application of our intracellular low-oxygen device "oxygen hunter" in scientific research, and he also provides a lot of ideas for our future iteration.
After the teacher's suggestion, we contacted Novi Credit again. They also expressed high appreciation for our project, affirming the value of intracellular low oxygen gene device in scientific research and enterprises, especially the application prospects and effect in enzyme preparation and biological nitrogen fixation.
After we entered the preparation link of the presentation, Mr.Yang Yu came to the group to listen to our complete presentation and report, and put forward some opinions on the narrative during the presentation, and taught us some tips and experiences of the report.
In terms of application, we contacted potential users and enterprises of the project - that is, experts and scholars in charge of relevant topics who may need to use the original hypoxia gene and R&D personnel of various enterprises. They all affirmed and supported the idea of our project, agreed to most of the possible application scenarios we envisaged, and of course, raised objections to a small number of application scenarios, helping us to deny these ideas. During the communication, they also mentioned some application ideas that we didn't think of at the beginning, and we have written these processes here.
In brainstorming, we considered the subject of biological nitrogen fixation. Although we could not continue because of many problems, it gave us inspiration for the current project. In the application part, we first considered the nitrogen fixation that we have considered. Through consulting the literature, we believe that the introduction of leghemoglobin into non symbiotic biological nitrogen fixation systems or some artificial nitrogen fixation systems can greatly improve the nitrogen fixation efficiency. First of all, we had a discussion with Xiaohong Zhou , who is engaged in nitrogen fixation project research in our school, on the feasibility of introducing leghemoglobin to promote nitrogen fixation, and got a positive answer. After that, we contacted Fengjiao Xin and other researchers of the Chinese Academy of Agricultural Sciences by email, who also gave their affirmation, and mentioned that they hoped we would do more research on leghemoglobin and try protein optimization to improve the combination of oxygen and help nitrogen fixation process.
Biological hydrogen production is also a topic that we had thought about when brainstorming. It is also an inspiration for our current project because some problems have not been completed. One natural hydrogen production path is that autotrophs such as green algae absorb light energy in photosynthesis to produce electrons, which are transferred to hydrogen production enzyme (hydrogen ase) to combine with hydrogen ions to produce hydrogen. However, when oxygen reaches a certain concentration, the activity of hydrogen producing enzyme will be significantly inhibited, resulting in the failure of continuous hydrogen production. We believe that the introduction of leghemoglobin and laccase to build an intracellular hypoxic environment can ensure the normal growth of organisms without inhibition of hydrogen producing enzymes, and achieve continuous and efficient hydrogen production under normal oxygen concentration. In terms of the background significance of the project, we had an exchange with Sun Kening, a teacher from the Institute of Energy and Chemical Engineering of our school who studied catalytic hydrogen production. The teacher recognized the advantages of biological hydrogen production in green and environmental protection, and discussed its efficiency with us. In addition, we also contacted Lili Xu, the author of a hydrogen production literature we found, and obtained her approval.
Biocarbon fixation is an application that we mentioned when communicating with COFCO Group and Honghui Pharmaceutical. This is because in order to achieve the goal of "carbon peak" and "carbon neutral" as scheduled in China, relevant environmental protection policies have been fully launched, and the government has given great support to relevant scientific research topics and green enterprises. Scientific research members of both enterprises believe that carbon fixation is also a reductive biochemical process restricted by oxygen, and our low oxygen system can be used to improve the efficiency of biological carbon fixation. We searched the literature according to their ideas, and found that the mainstream direction in the field of carbon fixation is to design artificial biological carbon fixation lines. It is often necessary to consider protein structure optimization to reduce oxygen sensitivity, but this will generally affect the efficiency of the carbon fixation lines they refer to, and our low oxygen system can solve this problem. We discussed this with Mr. Chun Li, who affirmed our idea and put forward some suggestions.
Synthesis of reducing drugs is a possible application field that we thought of at a group meeting after the topic was determined. With this idea in mind, we first consulted Jianhua Liang, a teacher in the pharmaceutical industry of our school. He believed that many reducing drugs did not achieve their goals because of this limitation, so most of them used chemical synthesis. If biological synthesis can be achieved, it will undoubtedly be more environmentally friendly. After that, we searched the literature in detail and found some drugs with strong reducibility. With the confirmation of Mr. Xudong Feng, we believe that during theβ- Carotene process, introduction of hypoxia system into carotene biological pathway can prevent its double bond structure from being oxidized and improve the yield.
Biological treatment of water body was also mentioned by our classmates at a group meeting. Some reports mentioned that biological nitrogen removal system is the most important method for deep nitrogen removal at present. So we also searched the literature and found the statement that denitrifying bacteria may need a low oxygen system. So we got in touch with a sewage treatment plant under Beijing ENFI Environmental Protection Co., Ltd. We originally planned to visit and discuss offline, but we had to do it online due to the epidemic. They are very positive about our idea, think that once industrialization can greatly improve the treatment effect, reduce industrial costs, and at the same time show the willingness to carry out pilot experiments, but unfortunately because of the epidemic, our experimental progress is very urgent, there is no opportunity to cooperate, and they hope to have more in-depth cooperation when improving the project next year.
While looking for possible downstream application scenarios, we also thought of some other schemes, including anaerobic fermentation of lactic acid bacteria, bread fermentation, biosynthesis of polyphenols, biosynthesis of aromatic amines, and biosynthesis of strong reducing vitamin C, but they were rejected one by one when communicating with COFCO. COFCO researchers told us that the current chemical methods of these schemes are very mature and have more or less problems in principle and industrialization.
During the school presentation, our teachers Minmin Liang and Jun Li mentioned that our hypoxic system may be used for targeted treatment of tumors, because the rapid growth of tumors will greatly reduce the oxygen around them, forming a hypoxic environment, which will enable cells to target the release of synthetic drugs near tumors. We haven't consulted the literature on this subject in detail, so we hope that we can practically evaluate the feasibility of this application scenario in the future.
In the process of brainstorming and topic selection, we once thought about using biofilm to actively transport seawater for desalination. There are two implementation methods.
One is the diaphragm method. A special cell with direction is designed through the synthetic biological method and embedded into the diaphragm, which separates the seawater in the container from the left and right. This kind of cells can absorb the salt in seawater from one side and discharge it on the other side, so that both sides can get fresh water and concentrated seawater respectively.
The other is absorption method, which is to design gene lines to enable cells to actively transport and absorb salt when receiving a signal, so as to desalinate the seawater around cells. When receiving another signal, the cells release the salt they just absorbed, thus realizing recycling.
But Hu Bing raised the problem of desalination. First of all, due to the damage of high salt to cells, cells generally do not transport high concentrations of ions into the cells, while those salt tolerant microorganisms simply expel or reject salt outside the cells, rather than being able to withstand the high osmotic pressure inside the cells. Secondly, seawater desalination needs to reach a certain standard. It can be used as fresh water only after desalination to at least 2% of the original salinity. It is very difficult to achieve such low salinity simply by relying on biofilm transport. Therefore, at the teacher's suggestion, the idea of desalination was rejected.
When selecting the topic of the project, we proposed biosynthesis of 3-methylthiopropanol to improve the efficiency of perfume synthesis, reduce its cost, and make it more widely available to thousands of households and serve the general public.
However, in the communication with COFCO, we stated this opinion to the experts, and then the experts, based on their production experience and knowledge level, rejected this idea.
On the one hand, the efficiency and cost-effectiveness of biosynthesis of 3-methylthiopropanol are still too low. The peak concentration of 3-methylthiopropanol is about 1.6 g/L at 40 h of batch fermentation, and the molar conversion rate is only 0.56 mol/mol, which is far less than other mature synthesis methods. With the extension of fermentation time, the mass concentration of 3-methylthiopropanol continues to decrease, indicating that 3-methylthiopropanol will be degraded or utilized by bacteria during biosynthesis. At the same time, as a spice with little addition, its application value is not particularly broad.
On the other hand, according to their understanding, nowadays, China has strict requirements for artificial food, and high-yield food additives obtained through transgenic means and methods are not allowed by law. Compared with foreign countries, as long as GRAS products, such as heme produced by beyond meat from yeast, can be considered as usable products. China has relatively low tolerance in this regard, so it is unrealistic for us to complete the promotion and industrialization of the project to improve the biosynthesis of 3-methylmercaptan in China.
Online Q&A between BIT-China Team and COFCO experts
In the brainstorming, we realized the importance of nitrogen fixation, and some students put forward the idea of biological nitrogen fixation. They wanted to use biological nitrogen fixation to promote green and safe production of crops, improve the fertilizer efficiency of biological nitrogen fixation, and completely or partially replace industrial nitrogen fertilizer. However, in further communication and discussion, as well as searching for relevant literature, we learned that there is still a lot of unknown space about the mechanism of hormone secretion by nitrogen fixing microorganisms and the molecular regulation of plant response to them. In addition, the anti stress and defense functions caused by nitrogen fixing microorganisms, how plants integrate these molecular signals, and how the plant immune system participates in them are also unknown; Whether nitrogen fixing microorganisms can induce the increase of plant root exudates, enhance the activity of plant nutrient transporters, and thus improve the efficiency of nutrient absorption and utilization when activating soil nutrients is considered to be solved; Moreover, due to the wide variety of Azotobacter, the growth promoting mechanism caused by its main functional bacteria and the molecular mechanism of its interaction with plants need to be further explored. Based on the above problems that still need to be further explored, we understand that when we choose this project, we will have a lot of problems that need to be studied and explored urgently. The research period for this project may exceed one year, so this idea is included in the stage to be considered.
When determining the topic, we have an alternative project - biological combined nitrogen fixation - saline alkali resistant nitrogen fixing bacteria and saline alkali land restoration.
Considering that the public and society attach great importance to bioethics issues, and the degree of trust in some experiments involving bioethics is not very high, we have made some questionnaires and advice on the biological combined nitrogen fixation experiment.
In the first questionnaire, we received a lot of concerns and doubts.
Nearly 33% of the respondents believe that our project is harmful to human health, 80% of the respondents believe that our project is harmful to the environment, including wildlife, 22% of the respondents believe that our project is harmful to materials, equipment and infrastructure, and 33% of the respondents believe that our project will break the norms of engineering biology.
We summarized that this problem belongs to the biosafety problem in bioethics by looking up the literature. Biosafety products involving synthetic biology may pose risks to human health and the environment, for example, synthetic viruses or bacteria are pathogenic to humans, or their products seriously harm certain kinds of animals or plants, disrupt the food chain and disrupt the ecological balance; Synthetic microorganisms may have unexpected interactions with the environment or other organisms, thus posing risks to the environment and public health; The release of synthetic microorganisms into the environment may cause gene horizontal transfer and affect the ecological balance, or generate abnormal functions due to evolution, resulting in unprecedented side effects on the environment and other organisms.
The introduction of our stress resistant gene into nitrogen fixing bacteria and its application in soil may have an impact on gene horizontal transfer and ecological balance, which may lead to variation of other flora in soil or other impacts, thus affecting human health. And our team did not come up with a way to prove that our practice is completely safe or reduce the risk. So based on this, we denied this project.
In the brainstorming session, we had the idea of using synthetic biology to treat hair loss, but we finally abandoned this topic because of the public's attitude towards it.
In recent years, hair loss has become more and more common. Hair loss has gradually become one of the health problems that people worry about. College students account for a considerable proportion of them. Hair loss can be divided into physiological hair loss and pathological hair loss. The existing research shows that, due to the periodicity of hair growth, 50-75 hairs per day are normal physiological hair loss, and more than 100 hairs per day for a long time are considered as pathological hair loss. If the hair loss is within the range of 100 hairs per day, but the conscious hair loss continues to decrease, it can also be diagnosed as pathological hair loss. In addition, if more than 50 hairs are lost in the morning, and more hair is lost when combing, it can also be judged as pathological hair loss. The purpose of this questionnaire is to investigate the current situation of hair loss among the public, explore the causes of hair loss among college students and the social problems reflected by it. The project idea of BIT China team is to investigate the public's attitude towards "using synthetic biology to treat hair loss".
Object
Using the method of random sampling, 359 people were selected from Liangxiang University Town, Fangshan District, Beijing as the research objects. A total of 359 valid questionnaires were collected, including 176 male students (49.03%) and 183 female students (50.97%). 86 freshmen (23.96%), 95 sophomores (26.46%), 158 seniors (44.01%), and 20 seniors (5.57%).
Method
The self-designed "Survey on the Problem of Hair Loss among College Students and the Public Attitude to Hair Loss Treatment by Synthetic Biology" was used to conduct the survey. The questionnaire were combined with single choice questions and multiple choice questions. The contents of the questionnaire included the survey on the current situation of hair loss among college students, the survey on the related factors of hair loss among college students, and the survey on the public attitude to hair loss treatment by synthetic biology.
Quality monitoring
On the basis of reading domestic and foreign literature related to hair loss, the questionnaire was designed according to scientific, objective and feasible principles, and relevant experts were consulted to demonstrate and modify the questionnaire. Before the formal investigation, the investigators shall receive unified training to ensure the smooth implementation of the investigation. During the survey, the investigator checked the mobile phone submission page on the spot after the students completed the questionnaire to confirm that the students completed the questionnaire and ensure the authenticity and effectiveness of the questionnaire.
Statistical findings
Export the survey results and establish a database using Excel. The data obtained were statistically analyzed with SPSS 22.0 software and Logistic regression analysis was used.
Single factor analysis of hair loss in college students
The survey results show that 79 (22.01%) college students "basically do not lose their hair", 280 (77.99%) college students have hair loss, and 166 (59.29%) of them have "physiological hair loss" (less than 100 hairs per day), 114 (31.75%) college students showed the degree of hair loss as "pathological hair loss" (the daily hair loss was more than 100, or the daily hair loss was between 50-100, and the hair loss was consciously reduced). The hair loss of college students was significantly different in gender, hair quality, frequency of shampoo, frequency of staying up late, emotional factors, diet preference, etc. (P<0.05), but not in grade and major (P>0.05).
Multifactor Logistic Regression Analysis of Hair Loss in College Students
"Physiological alopecia" and "pathological alopecia" both take basically no alopecia as a reference, and take statistically significant sex, profession, hair quality, hair washing frequency, staying up frequency, and anxiety level as independent variables in single factor analysis to conduct logistic regression analysis. The results show that gender and staying up frequency are the influencing factors of physiological alopecia (P<0.05), and gender, hair washing frequency The frequency of staying up late was the influencing factor of pathological alopecia (P<0.05).
A survey of public attitudes towards hair loss treatment with Hechen Biology
After ensuring that the respondents fully understand the idea and principle of "targeted hair loss treatment with magnetotactic bacteria" designed by us, we asked them to rate the feasibility and safety of this project. Among them, 83 (23.12%) chose 0 point (totally distrustful of the scheme), 192 (53.48%) chose to give 4 points (believing that the project is of certain value, but will not take the initiative to try), 68 (18.94%) chose 7 points (believing that the project is meaningful, but will not necessarily try), and 16 gave 10 points (believing that the project is meaningful, and willing to try). The final average score is 3.91, which reflects that the public has some doubts about the feasibility of the project itself, and has a great concern about the safety of the project, "unwilling to take the initiative to try".
At the same time, we also investigated the specific opinions and ideas of the public on our project. Among them, 186 (51.81%) students thought that there were relatively mature hair loss treatment schemes on the market, such as anti hair loss shampoo, hair transplant and hair receiving technology, and that the project was of little significance; In addition, 264 (73.54%) students worried that the treatment cycle of this scheme was too long, or the treatment price was unbearable; In addition, 189 (52.65%) students still have doubts about the expected effect of the project. Of course, the most worrying thing for the public is still the side effects of this treatment scheme. 294 (81.89%) students were worried that this would have a bad impact on other parts of the body. Safety is still the primary consideration for people to choose a scheme.
Conclusion
The results showed that 280 (77.99%) college students had alopecia, of which 59.29% were physiological alopecia and 31.75% were pathological alopecia. It can be seen that hair loss is widespread among college students, and many college students have relatively serious hair loss, which has developed into pathological hair loss. Although college students are not the high-risk group of hair loss, it is undeniable that hair loss has become one of the health problems faced by college students, which should be paid extensive attention.
The inducements of hair loss of college students are mainly manifested in two aspects: work and rest habits and emotions. Irregular work and rest and bad emotional factors have a great impact on the problem of hair loss of college students. Among the people with pathological alopecia, "often staying up late" accounts for the highest proportion. Frequently staying up late will disorder the endocrine system and immune system, causing scalp hair follicles to produce a toxin, reducing the vitality of scalp hair follicles, and then causing hair to fall off in large quantities. But everyone's immune mechanism and reaction are different, and not everyone will suddenly lose a lot of hair. As for a few students who do not stay up late but still have severe alopecia, it may be related to insufficient nutrition in their daily diet or genetic factors, such as congenital alopecia, alopecia areata and androgen derived alopecia caused by genetic problems such as genes, growth factors and cytokines.
At the same time, our survey shows that the public is worried about the treatment scheme of pathological alopecia using synthetic biology, and few people are willing to take the initiative to try this scheme for treatment. The public's concern about the project is focused on the fact that there is a relatively mature treatment scheme for hair loss on the market, and they think that the project is of little significance; Concerned that the treatment cycle of this scheme is too long, or the treatment price is unbearable; Doubt about the expected effect of the project, side effects, etc., will bring bad effects to other parts of the body. Based on this, we come to the conclusion that the public has insufficient confidence and trust in the use of synthetic biology for hair loss treatment. The reason is concentrated on the safety, cost and effect. The idea of trying to use magnetotactic bacteria to treat hair loss is not yet mature and the public acceptance is low.
In the principle design, we found Mr.Yang Yu, and had an in-depth communication with him on the experimental program mainly for laccase and leghemoglobin.
Laccase contains copper ions, and its mechanism of action is actually that oxygen oxidizes the monovalent copper in the center of the laccase, and then the bivalent copper takes over the electrons in the organic matter, thus circulating to realize the reaction of oxygen and organic matter. Laccase does require the addition of copper ions to the culture medium when expressed. In the past, laccase is expressed and extracted and purified before copper ions are added. However, if it is necessary to be added in the culture medium of cultivating bacteria, the concentration of copper ion should be reduced. Too high the concentration will inhibit the growth of E. coli and even cause its death. For our previously proposed adding 1mM copper ion to the culture medium, the teacher thought that the high concentration would cause significant inhibition on the growth of E. coli. Based on the teacher's suggestion, we adopted the copper ion concentration of the copper ion concentration of 0mM, 0.2mM, 0.5mM in the subsequent experimental design, specifically by adding 1ml of a specific concentration of copper sulfate solution to the culture medium.
leghemoglobin is also an area that the teacher was exposed to before, and Yu's chosen method was to test the hydrogen production of E. coli. As our brainstorming information suggests, Escherichia coli can also produce hydrogen as a heterotroph, and its hydrogenic enzymes are also sensitive to oxygen. The teacher affirmed that our use of oxygen-sensitive promoters were connected to the fluorescent protein test method, believing that it is a good high-throughput detection method. We told the teacher about the idea of adding hemin to the medium to supplement the heme. The teacher's suggestion was that the large hemin molecules are likely unable to enter the cells. It is also proposed that some precursors of heme, such as γ -aminobutyric acid (ALA), can can cross the membrane. In fact, E. coli does not synthesize hemoglobin, but does heme. This is because E. coli also requires heme iron to regulate its redox metabolism through bivalent versus trivalent changes. This suggests that E. coli can autonomously synthesize the heme, which allows us to increase our confidence, although there is no guarantee that the heme can be adequately supplied to the leghemoglobin. The teacher mentioned that leghemoglobin can carry oxygen, but the oxygen concentration reaches a certain amount, and the oxygen transport capacity is no longer significant. Therefore, the original oxygen has a lower concentration. In this regard, the teacher suggested that we use laccase and leghemoglobin, first use laccase to reduce the oxygen concentration, and further reduce the oxygen concentration.
In the use of the experimental device, we also adopted the opinion of Teacher Yang Yu. Because the testing module requires low oxygen cultivation, senior Ling Hao in the teacher group is doing related topics, and they suggested that we use a custom-sized culture bottle to achieve oxygen extraction. But because of the epidemic reasons, we lost contact with the original order manufacturers (I hope they did not close down due to the epidemic reasons), so we replaced this method with nitrogen filling gas.
For the problem we encountered that PCR could not obtain the desired fragments, we consulted —— GENEWIZ, the company that synthesized genetic primers.
They responded with three reasons to consider:
1. Reasons for the experimental materials, such as ensuring that the quality of the template, primers, PCR enzyme, and working solution is no problem;
2. The sequence itself may be difficult to amplify, such as high GC content, low GC content, hairpin structure, repeat structure will lead to PCR difficulties, must be more repeat amplification;
3. The sequence TM value of primer and template binding should be controlled between 40℃ and 60℃, and the annealing temperature during PCR should be set accordingly according to the GC content of the sequence.
Through this exchange, we have been inspired to some extent. In the subsequent trials, we will find more suitable primers, and try several conditions during PCR, to find the most appropriate conditions, and thus improve the success rate of the experiment.
When our project was possibly related to the pharmaceutical industry, Mr.Liu Hu introduced Dr. Li Qiaofeng from Honghui New Pharmaceutical Technology Co., Ltd., which we contacted immediately. On the afternoon of September 13th, we had an online communication with Beijing Honghui New Pharmaceutical Technology Co., Ltd.
We learned that Beijing Honghui new medical technology co., LTD. Is in 2002 by a Swedish returned students entrepreneurial team founded in Beijing zhongguancun pharmaceutical research and development of high-tech enterprises, the company is located in daxing district biological medicine industry base, is currently a wholly owned subsidiary of exhibition health, is a focus on medical science and development of international research and technology innovation enterprises. Since its establishment, the company has been recognized as a high-tech enterprise in Beijing, and its main business is customized synthesis of small chemical small molecules. In addition, the company does not stop here and expand more aspects, such as research and development of intermediates for clinical research, analysis and testing services, construction of drug activity screening platform and so on.
Meeting screenshots with Honghui
At the meeting, Dr. Li Qiaofeng mentioned that the company's independently developed drugs for stroke treatment with brain nerve protection, as well as drugs to improve the protection rate of patients' brain injury in a low oxygen environment, mainly to deal with altitude sickness. Again, Dr Li Qiaofeng began to talk about his work: mainly for the transformation of enzyme molecules, the first is to find the right enzyme, and then the enzyme molecule molecular modification to improve the activity of the enzyme, and then the application of E. coli a large number of culture strains after isolating enzyme enrichment, finally with the reaction of enzymes and substrate to get the final product.
Afterwards, we introduced Dr. Li to our project, including background inspiration, experimentation, modeling, and possible future applications. Dr. Li affirmed our idea of the project. After further exchanging the design ideas of function modules and detection modules, he thought that there was no problem with the project in principle. Finally, he put forward some suggestions for our application.
Here are our specific exchanges:
Online Q&A between BIT-China Team and Honghui New Pharmaceutical Technology Co., LTD
In September 2022, representatives of BIT-China team, accompanied by instructors, visited COFCO Nutrition and Health Research Institute in Changping District, Beijing, and under the leadership of senior engineer Wang Xiaoyan, communicated with senior researchers Zhou Nana and Zhang Yuan, and gained a deeper understanding of the project.
The researchers first briefly presented and introduced COFCO. Founded in 1949, COFCO Co., Ltd.) is the largest grain, oil and food enterprise in China and a leading diversified product and service supplier in the fields of agricultural products and food. It is committed to building grain, oil and food enterprises in the whole industry chain from field to table, and building an urban complex in the full service chain. Make use of constantly renewable natural resources to provide nutritious and healthy food, high-quality living space and life services for human beings, and contribute to people's life of prosperity and social prosperity and stability. The COFCO Nutrition and Health Research Institute, a subsidiary of COFCO, is the first domestic research and development center to conduct systematic research on the nutritional needs and metabolic mechanisms of the Chinese people to realize the Chinese people's health demands. COFCO is a large group with many research institutes and listed companies, covering a wide range of businesses, including cooking oil, drinks, staple food, seasonings and so on.
After we arrived, we held an exchange meeting with COFCO researchers. At the meeting, we first introduced the simple situation of this year's iGEM project. We described the inspiration of the project, and then we introduced the methods of function modules and detection modules in the experimental design, which won their recognition. Finally, we looked forward to the possible prospects of the project, such as the production of substances easy to oxidation, and they helped us exclude some difficult applications.
We visited the cofco nutrition and health research institute, briefly understand the institute of the National Energy Administration of biological liquid fuel research and development (experiment) center, cofco biochemical technology research and development innovation center in Beijing base, biological fermentation innovation studio and biotechnology center four research center, and has carried on the exchange and cooperation, produced a new understanding.
visit to COFCO Nutrition and Health Research Institute
Senior engineer Wang Xiaoyan first took us to visit the National Energy Administration of biological liquid fuel research and development (experiment) center, we learned from the establishment of the historical module exhibition hall, "national energy biological liquid fuel research and development (experiment) center" aims to build an innovative country, promote the energy structure upgrading and energy science and technology progress, promote the joint, technical research in the field of biological liquid fuel, promote industrial scientific and technological progress and the sustainable development of biomass energy industry. The R & D center is committed to the development of biomass energy, with the development of non-food alternative fuel ethanol and the use of cellulose as the key development direction. And undertook a number of research and development tasks, focusing on solving the cellulosic ethanol storage and transportation equipment, improving the cellulosic ethanol pilot base.
The research center focuses on biofuel ethanol-related modules, mainly involving four aspects: (1) the development of new functional yeast. Independent development of glycan saccharide yeast and high temperature resistant yeast, so that the annual saving of 6.5 million yuan of saccharidase cost and nearly ten million frozen water and electricity consumption.(2) Diversified raw material for ethanol production technology. We solved the problems in producing alcohol from all rice, wheat and other raw materials, developed collinear ethanol production technology from multiple raw materials, and helped process 7.1 million tons of old rice, 1.9 million tons of brown rice, and 680,000 tons of old wheat to ensure food security.(3) Alcohol fermentation and production technology of thick mash. The alcohol fermentation technology of intermittent thick mash and continuous thick mash was developed. The wine content reached 17.3%VOL and 15.3%VOL respectively, with the annual cost reduction and efficiency increase of 23.2 million yuan. The continuous thick mash technology has reached the international leading level.(4) Cellulosic ethanol production technology. Independent development of a complete set of cellulosic ethanol technology, to solve the problem of "bottleneck", the overall technology reached the international advanced level.
By visiting the Bioliquid Fuel Research & Development (Experimental) Center, we have learned that there are some key enzymes and intermediates that require low oxygen to be better effective and reduce the production of by-products. In the production process, some auxiliary reducing agents may be used to reduce the oxygen effect. However, if our intracellular hypoxia technology can be introduced, it can indeed simplify the experimental procedures and improve the production efficiency to some extent.
We came to cofco biochemical technology research and development innovation center in Beijing base, first visited the enzyme catalytic pilot production line, the base has a set of advanced unit equipment, including enzyme catalysis, continuous ion exchange system, ultrafiltration, nanofiltration membrane system, vacuum concentration system, crystallization system, such as pilot plant, pilot production capacity of hundreds of tons, completed the GTP, CTP, UTP and ATP products such as trial production. Then we visited the fermentation pilot production line, with a new 30L / 300L / 3000L step by step amplification of the pilot fermentation system and many other chemical production research devices. The pilot test of nucleotide series products has been completed, and the annual production capacity reaches hundreds of tons, among which the enzymatic unyield rate of UMP, CMP, GMP and AMP fermentation process has reached more than 85%.
By visiting the Beijing base of COFCO Biochemical Technology Research, Development and Innovation Center, we learned that if the low oxygen system of our project can be used in the process of enzyme catalysis and membrane filtration, the activity of key oxygen-sensitive enzymes in cells can be maintained, reduce the interference of oxygen to some experiments, and reduce the production of reactive oxygen species. This greatly improves the operability of the production of some compounds, and also improves the stability of products during preservation. In this way, there is no need to install external low oxygen devices, but also can make cells grow and reproduce efficiently, with a wide range of application scenarios. But it may be a long way to go to fully optimize the device and achieve industrial application.
Visiting COFCO Biochemical Technology R & D and Innovation Center in Beijing base
Through the explanation of the staff, we understand that the fermentation process is a small experimental research to industrial production transition, the basic completion on the small test synthesis to workshop production process, to ensure that the process procedures can always produce predetermined quality standard of qualified products, we think of our project is just stay in the laboratory experiment, if you want to apply our theoretical research to large factories also need to consider more aspects, also need to face more tests.
We enter the biological fermentation innovation and efficiency studio to visit cofco fermentation industry, biological fermentation industry is one of the key development of cofco strategic emerging industries, in order to better cater to the industry development, power biological fermentation related enterprises to grasp the market opportunities and challenges, will provide the whole industry chain for biological fermentation online international trade negotiation space, for biotechnology industry innovation and development, to meet the biological industry new blue ocean. We also saw the samples of many strains displayed by COFCO, such as Clostridium butyricum, Lactobacillus plantarum, etc. The staff told us about the life course and anaerobic mechanism of these anaerobic bacteria, which inspired us to construct a hypoxia environment.
By visiting the biological fermentation innovation and efficiency studio, we have learned from experts that the anaerobic device designed by our project is promising in genetic engineering. For example, the functional genes of anaerobic bacteria can be introduced into the aerobic bacteria, and cooperate with other elements, to complete some novel tasks, and reduce the difficulty of anaerobic and aerobic bacteria gene reorganization. We also communicate with the experts about some operational problems in the construction of synthetic biological systems, such as the methods of building plasmids or importing genes.
Finally, we came to the Biotechnology Center of the Institute of Nutrition and Health. The laboratory has a comprehensive research platform ranging from synthetic principles of biology, engineering bacteria research, biological separation process development to final production process research, as well as a digital platform for modeling problems and process analysis. In addition, the biotechnology center has little research on functional sugars, bio-based materials and other aspects. The technology center has independently designed to prepare aloxenone sugar with high conversion rate and high stability differential isomerase and supporting purification process; mutagenesis and high-throughput screening of erythritol synthesis, etc. These functional sugars have the functions of blood glucose control and dental caries prevention, and are widely used in food, daily chemicals and medicine. The biological base materials polyhydroxyfatty acid ester (PHA) and polylactic acid (PLA) have achieved high efficiency mass production, committed to packaging materials, fiber textile, biomedicine, agriculture and other fields.
At the same time, the staff led us to visit the laboratory of COFCO Biotechnology Center. We also saw some advanced equipment, such as large centrifuges, fermenters, and so on. And under the introduction of the researchers to understand its use, mastered a lot of usually in the school laboratory difficult to contact the novel knowledge. Among them, there is a set of automatic large sampling device, which can realize high-throughput sampling and high-throughput detection in a short time, which can greatly improve the efficiency. The official told us that if we need experimental equipment, we can apply. The teacher in charge of the instrument will lead us to use the instrument and assist us with the required experiments. The researchers and teachers from COFCO Biotechnology Center gave us recognition and help. They also talk about the model of research and development in enterprises, as well as some differences between enterprise research and development and university research and development.
By visiting the Biotechnology Center, we learned that during the production process, some environments requiring low oxygen do occur, and they may also be used for our projects later. However, because the project is inclined to be basic, there is still a long way to go to make the engineering fungus belt. When engineering, we need to consider the market-related issues that laboratory products need to pay attention to to achieve revenue in the industry, so as to find a foothold in the market.
A photo of us
Finally, our team had an in-depth exchange with the experts of various research institutes.
Below: Q: BIT-China. A: China Oil and Food Import and Export Corporation
BIT-China team and COFCO experts exchange and consultation Q&A
Introduction to the project
Before we started the task, we basically decided to use MATLAB as our modeling software. Because some team members are not familiar with the use of the software, in order to ensure the orderly development of the later modeling work, we carried out the learning and training on the use of MATLAB software during the winter vacation. We used the resources on the BiliBili website. Among the courses of the University of Science and Technology of China as the traininnumerous rich curriculum resources, we finally decided to choose the open g materials. The students in the modeling group watched the teaching videos together and practiced using MATLAB, laying a foundation for the stable promotion of the subsequent modeling process.
Construction of oxygen diffusion model based on Fick's diffusion law
Since most of the members of the modeling group are not biochemical students, in addition to communicating and learning the biochemical knowledge related to Fick's diffusion law with the students of biochemistry, we also supplemented the relevant content with the help of Zhihu and other platforms, which greatly enhanced the understanding of Fick's diffusion law and broadened our horizons. Under the condition of fully understanding the contents of Fick's diffusion law, our modeling group, led by Li Yang, first deduced the differential equation of the basic diffusion model on the draft paper, and completed the first goal of the task.
The modeling team communicates with bloggers on the Zhihu platform on their understanding(Translated)
After the construction of oxygen diffusion model, we began to consider the oxygen consumption of cell biochemical reaction. When considering the oxygen consumption of the biochemical reaction on the membrane, we determined the main biological activities that affect the oxygen concentration. First of all, we determined the differential equation of oxygen consumption rate in each biological activity (except leghemoglobin) through the study of biological knowledge. When considering the oxygen consumption of intracellular biochemical reaction, we focus on the oxygen consumption of laccase and leghemoglobin. The differential equation of oxygen consumption rate of laccase was quickly summarized after consulting relevant data. We determined the parameters of each differential equation through experimental measurement and literature review, and completed the corresponding model construction. Since most of the members of the modeling group do not have much modeling experience, after listing all the differential equations, we encountered difficulties in selecting solutions. Through CSDN, we learned about the dsolve function and solver class function. Considering that our equations involve more than 30 variables and more than 200 parameters, we agree that it is difficult to guarantee the existence of the exact solution of dsolve. When selecting solver class functions, the scope of application of each function makes it difficult to select. After further searching in CSDN, we found that there was a blog about the solution of dynamic differential equations, in which two solutions, ode45 and ode15s, were recommended. After trying, we decided to use ode15s as the final solution of our dynamic differential equations.
The modeling team communicates with bloggers on the CSDN platform about the solution selection of differential equations(Translated)
Because we could not find the relevant content in the process of oxygen consumption of leghemoglobin through reading the literature, we finally decided to predict the oxygen consumption parameters of leghemoglobin through BP neural network. As for how to build BP neural network, almost everyone in the modeling group has no relevant experience, so we turned to CSDN and Github again. Finally, in a CSDN blog introducing the steps of building BP neural network with matlab, we specifically understood the structure and principle of BP neural network, and finally selected Levenberg Marquardt (gradient descent algorithm) as the training function and sigmoid (sigmoid) as the activation function to complete the building of BP neural network model. In addition, we found the reference code of the corresponding function on Github. After reading the relevant content of CSDN and GitHub to learn and understand BP neural network, we finally completed the construction of BP neural network model and realized data processing and prediction by collecting a large number of data collected in experiments and literature review.
The modeling group communicates with bloggers on the CSDN platform on the selection of BP neural network activation function(Translated)
In order to complete the establishment of BP neural network model as soon as possible, during the process of waiting for data collection, Group Leader Li Yang used the resources on CSDN and Github to compile a small matlab application of BP neural network, and improved the design and construction of its GUI, which shortened the time consumed for the establishment of the entire model. Finally, we sorted out and summarized all the models and completed the expected objectives of the modeling team.
After completing the basic tasks, we released the demonstration of the results of the modeling work and the explanation and science popularization of part of the modeling process on the Bilibili website, and received a considerable amount of praise and feedback. Some comments provided us with valuable suggestions for improvement, which benefited us greatly and helped to promote the subsequent improvement work.
In the process of team modeling, the application of MATLAB is an essential and crucial link. Although the students in the modeling group have mastered the basic functions of MATLAB in their daily study, it is still a very difficult task to apply MATLAB to scientific research and engineering calculation. So in order to apply MATLAB to our project and simulate our experimental principles and experimental projects, the students in the modeling group who are majoring in optoelectronics contacted their head teacher, Mr. Liu Xianwen from the School of Optoelectronics of Beijing Institution of Technology, hoping to learn some knowledge about MATLAB in engineering calculation from him.
Considering that our project is to simulate the dynamic equation of laccase and leghemoglobin consuming oxygen, in order to simulate and solve this complex dynamic equation set, we asked Mr. Liu how to simulate the complex dynamic differential equation set on MATLAB. Under the guidance of Mr. Liu, we learned the method of ode to solve differential equations, and learned different series of differential equations corresponding to ode series functions. By further searching the literature, we decided to use ode15s function to solve the complex continuous dynamic differential equations.
In addition, in order to realize some prediction of the dynamic coefficient in the project, we are exposed to the neural network prediction method. But this knowledge is strange to us. Therefore, we asked Mr. Liu for some knowledge about neural networks on MATLAB. With the help of Mr. Liu, we learned that MATLAB has its own neural network toolbox, which can complete neural network prediction, clustering analysis, time series prediction, etc. However, because the specific algorithm of neural network is complex, it will take a long time to master it, so Mr. Liu does not suggest that we learn the algorithm of neural network in detail. So from the perspective of engineering, after consulting the help documents of MATLAB and reading the relevant materials of MATLAB community, we chose to directly use the relevant neural network functions and neural network toolbox provided by MATLAB.
However, in the operation of the actual model, due to the large amount of data and many solving processes, the operation time of the model is very long. Because we don't know much about solving differential equations in MATLAB, we don't know how to deal with this problem for a while. Finally, by seeking the help of Mr. Liu again, we improved the problem that the solution time was too long. In addition, Mr. Liu suggested that we could consider changing the algorithm for solving the equations and the division accuracy, so as to reduce the calculation time. Under the patient guidance of Mr. Liu, we learned and mastered how to solve differential equations with MATLAB from scratch, and how to improve our specific solution program in engineering applications.
Feedback from netizens after the modeling group released the model content in Bilibili(Translated)
After we preliminary analyzed the experimental data results, we communicated with Professor Yu Yang of our school with questions. First, we showed our data results and curves to Professor Yu Yang and discussed with him. He thought that our experimental data could basically prove that the low oxygen device such as leghemoglobin and laccase could achieve a certain degree of reduction in oxygen concentration. He also agreed and approved most of our analysis of experimental results. For example, the previous experience with laccase in his research group showed that although laccase promoted aerobic metabolism differently from leghemoglobin in principle, it did have little effect on the growth of E. coli. In addition, he was surprised by the analysis we found through literature that when both laccase and leghemoglobin were introduced, the copper ion of 0.5mmol/L had the best effect on oxygen consumption -- high concentration of copper ion would help the expression of leghemoglobin. At the same time, professor Yu also questioned some of our analysis and raised his own ideas. For example, he thought that the reason why the expression of hemoglobin in the group without heme had the best effect was that heme might react with oxygen and copper ions, affecting the concentration of oxygen.
In addition to the result analysis, Professor Yu also supplemented and optimized some existing ideas in the iteration aspect of our project and provided some new ideas. In the end, she also gave us some valuable opinions on the narrative expression of the project and carefully taught the students who were going to participate in the presentation.
A joyful group photo of BIT-China and Professor Yu Yang after communication
We first asked questions with Professor Zhou Xiaohong, who is engaged in nitrogen fixation project research, about the feasibility of introducing leghemoglobin to promote nitrogen fixation.We got a positive answer from Professor Zhou. He believes that our project has high feasibility and sufficient practical significance, and has answered and discussed some questions related to our project.
Beijing Enfei Environmental Protection Co., Ltd., Wang ichao, water treatment engineer, enthusiastically answered our question
