Team:FAFU-CHINA/Human Practices
Overview
During the inspiration phase of the project, we investigated the whole flavor industry. Found that the whole
world for flavor demand is increasing year by year. There are very few terpenes in plants. Can't meet the
growing market demand. Chemical synthesis methods produce more by-products and may cause pollution. With the
development of synthetic biology, the production of spices by microbial fermentation has a broad prospect[1].
Spices are widely used in daily chemical, food and medical fields. Different aroma substances have different
effects on human experience. For example, let the mood feel happy, lower blood pressure, sterilization and
disinfection. After several rounds of investigation. We finally identified linalool and neroli as our final
products.
Safe Supporting
In order to learn more about issues related to informed consent, privacy and data protection, and promote the
better implementation of human practice in our team. We contacted Professor Liye Zhan of Fujian Agriculture and
Forestry University. Professor Liye Zhan has been engaged in mental health education for many years, has rich
experience in psychological counseling, has participated in human social practice activities for many times, has
certain opinions on social ethics knowledge and personal privacy protection, and pays attention to summarizing
experience. In Journal of Southwest University for nationalities, Chinese school health More than 20 papers have
been published in various core journals such as China Journal of mental health and higher agricultural
education.
We asked Professor Zhan: "How can we better show the results we want while protecting the privacy of the
interviewee ?" Professor Zhan suggested that we should let the interviewee sign the informed consent form and let
the interviewee know the purpose of our interview.
We have adopted the opinions of the professor. When collecting the questionnaire, we will carefully explain our
intention to the masses. The questionnaires are conducted anonymously. Nevertheless, we will ask the respondents
whether they are willing to publish the questionnaire results.
Then we asked, "we often go to other schools to give lectures for some students. What do we need to pay
attention to ?"
The professor asked us to report to the relevant departments and get their permission before giving a speech in
other schools. At the same time, we should pay attention to protecting the privacy of minors and not disclose
their personal information on the Internet.
We agree with the teacher very much. Before carrying out education and publicity activities, we obtained the
consent of instructors and relevant institutions, and carried out relevant activities under their guidance. On
the other hand, the photos taken on site can only be used in Wiki after being approved by the relevant person in
charge and the relevant guardian. We didn't disclose any personal information on Wiki except photos.
In view of the ethical problems existing in the investigation and research, Professor Zhan hopes that we will
seriously study the relevant legal knowledge. Carry out social research activities on the premise of fully
respecting personal privacy and personal wishes.
We listened to the teacher's suggestions and carefully studied the relevant laws and regulations. In the future,
I hope we can carry out human practical activities on the premise of legality and morality.
Problem investigation
Ye Ai
Associate professor of garden college and Art College
Research interests: garden plant breeding and cultivation,garden plant biotechnology,garden plant
application
We asked the teacher some questions related to fragrance, and we wanted to know the factors related to the aromatic substances produced by plants.
Q: In the breeding process of orchids. What factors are generally related to the fragrance of orchids ?
A: From the perspective of endogenous factors, the floral aroma of orchid is generally related to the aroma
genes it carries, but it is related to its growth environment, soil types, and nutrients absorbed during its
growth. Therefore, the flower fragrance produced by orchids is related to its variety and growth environment.
Q: What factors are related to the expression of flower aroma genes ? A: We all know that gene expression is
inseparable from time and space, so the expression of the flower aroma gene is no exception. It is also related
to climate and geographical location. But most fundamentally, it is the type of gene that determines the type
and concentration of aroma.
A: We all know that gene expression is inseparable from time and space, so the expression of flower aroma gene
is no exception. It is also related to climate and geographical location. But most fundamentally, it is the type
of gene that determines the type and concentration of aroma.
Q: What are the aroma components of flowers and plants, and what is their content in flowers ? What impact will
these flower fragrances have on the human body?
A: Flower fragrance is composed of a
variety of chemical substances, such as some volatile organic acids and terpenoids, which are rarely synthesized
by plants. The reason why humans can perceive these fragrances is that the human sensory threshold for these
substances is low. Plants synthesize these aromas to attract bees and drive away pests. When people inhale these
aroma substances, these substances play a role in the human body,
which can play the role of sterilization, insect repellent, sterilization, and even disease treatment.
Summary: Floral fragrance is related to many physical and chemical factors, and the external environment
will
affect the level and quality of aromatic substances produced by plants. The floral fragrance is mainly produced
by volatile organic acids and terpenoids. We will follow this clue to
find the target products of the chassis organisms.
Yuting Tian
professor,doctoral supervisor,Fujian Agriculture and Forestry University，School of Food Science
Research fields:
primary processing technology and equipment of agricultural products, mining and
development of traditional fermented food resources, research and industrialization of animal and plant
functional food.
We introduced professor Tian to our idea of using microbes to produce spices, and we also introduced the
teacher to synthetic biology. We would like to inquire about the feasibility and safety of using
microorganisms to produce spices.
Feedback :The teacher pointed out that the use of fragrance additives should take safety into consideration first. Specific requirements should refer to national standards. Adding too little flavor may not have an obvious effect, adding too much flavor may affect the abuse of flavor substances will cause harm to the human body. This is not because of the by-products of chemical synthesis but because of some toxicity of the aroma itself. Or they might react with the food itself. Reduced nutritional value. In the meantime, certain aroma materials can cause the immune response of the human body, namely the allergic reaction that we often say. Therefore, flavor additives must be added within a certain range.
Improvement: We intend to control the copy number of each rate-limiting enzyme in the MVA pathway, thus controlling the microbial production rate of flavor. Make sure that household products (such as microbial night lights) do not produce high levels of aromatic substances that could adversely affect the human body.
Weiyi Zhang
Associate Professor, College of forestry, Fujian agriculture and Forestry University
Research interests: Research on forest health benefits, aromatherapy of plant essential oils,
environmental education and environmental literacy assessment, community forestry and ecotourism.
During the preliminary design of the project experiment, the team visited Associate Professor Zhang
Weiyin of the school of forestry of Fujian agriculture and Forestry University. Professor Zhang is
engaged in the research on the health benefits of forest health, aromatherapy of plant essential oils,
environmental education and environmental literacy assessment, and teaching and scientific research in
the field of community forestry and ecotourism. We brought ionone, citral, citronellal, nerol and
linalool to interview the teacher. I hope that the teacher will introduce us to the effects of various
spices on the human body and recommend suitable spices for our project.
Feedback: The professor expressed his appreciation and affirmation for the team's project and led the team to visit his Forest Therapy and Detection Lab. At the same time, the teacher suggested that we choose spices with a mild smell as far as possible, and control the concentration of fragrance, so as to avoid too strong or too exciting fragrance will make some people feel uncomfortable. On the other hand, the teacher suggested that we start with simple substances so that there is no need to make major changes to living things.
Improvement: We finally chose linalool and nerol as our products, because their smell is easier to accept and easier to biosynthesize.
Xingtan Zhanng
Associate Professor, genome and Biotechnology Research Center, Fujian agriculture and Forestry University
Research interests: plant functional genomics research, including the assembly and typing of complex genomes, the development of Bioinformation Analysis Technology, and the mining of important functional genes based on omics big data.
We hope to theoretically verify the feasibility of BBa_K3732000 through the form of protein modeling, so we asked Mr. Zhang about the related methods of protein modeling.
Feedback： After understanding our project and modeling scheme, Zhang suggested that we conduct a comprehensive analysis of LOV2 and j based on the latest existing protein ab initio prediction modeling literature and the recently introduced hot alphafold2 algorithmα. The spiral allosteric structure is simulated and evaluated under light-dark conditions, and the scoring mechanism is adopted. The one with the highest score should be the best model choice. He believes that there are two difficulties in modeling at this time: time cycle and modeling challenges.
Improvement：We listened carefully to his suggestions with an open mind. We use methods such as Alphafold2 to comprehensively analyze LOV2 and its Jα helix.
Yong Jiang
Professor of School of Computer and Information
Research direction:
Research and teaching of analysis of multiple complex variables and mathematical modeling.
Q: What do you think is the role of mathematical modeling in the direction of biology ?
A: Mathematical modeling is applied to all aspects of life. As long as mathematical modeling is used reasonably
and effectively, it will also play a prominent role in the biological field.
Q: In the mathematical modeling of this project, our equation is a first-order differential equation. What
details need to be paid attention to ?
A: First, it is necessary to clarify whether the first-order differential equation is a linear equation or an
ordinary differential equation; secondly, after clarifying the specific type of the equation, consult the
relevant books to obtain the accurate code; finally, after fully learning and mastering the code, use MATLAB to
proceed Programming solution.
Q: If there is an error in the process of solving the differential equation, what do you think may be the
cause ?
A: It may be the problem of the differential equation itself, it may be an error in the code syntax, or it may
be a problem with the MATLAB version. When using MATLAB, you need to verify it many times to make the solution
result as accurate as possible.
literature search
Linalool
Natural linalool smells pure, round, sweet, elegant, with similar lily of the valley, lily pattern aroma. The global annual consumption of linalool is more than 1 000 t, which is mainly used in cosmetics, household detergents, furniture care products, pesticides, fragrance manufacturing, food and beverage processing, linalool is also used as a flavor and flavoring agent. Linalool is an important intermediate in the synthesis of vitamin E in the industry[2].
The InternationalJoint FAO/WHO Expert Committee on Food Additives (JECFA)established an Acceptable Daily
Intake (ADI) of 0–0.5mg/kg body weight/day for linalool.We hope that our microbial aroma production
the system can meet this standard and control the efficiency of aroma production to a level that will not
negatively affect biology and the environment.
We also noted the medical and health applications of linalool, which has analgesic, anti-anxiety,
anti-inflammatory and antibacterial effects[3].
With the rapid development of modern society, more and more people suffer from anxiety disorders of
different degrees. Anxiety disorders can be treated by drugs, the commonly used treatment drugs for
benzodiazepines, carbamate, diphenylmethane, and other chemical drugs, but these drugs have certain toxic
and side effects, long-term use will also lead to drug tolerance, drug dependence, and addiction. Linck
et al. confirmed that mice inhaled linalool showed anti-anxiety activity in light and dark test,
improved social behavior and reduced aggressive behavior, and only at high dose, the side effect of
damaging memory function would appear[4].
This is due to the neuroregulatory mechanisms of glutamate and GABA, which is similar to the FZU-China
team's project. So we got in touch with their team. And interviewed experts in related fields. Finally,
we clarified the regulation mechanism of linalool on the central nervous system. It provides a theoretical
basis for our products to be put into the market[5].
Neroli
Neroli alcohol has the fragrance of rose and neroli, which is a valuable spice and widely used in the
preparation of food, food, and the high-end essence of daily chemicals. Neroli alcohol is the edible spice
allowed to be used in China gb2760-2014.
In early social studies, many people reported to us that linalool didn't smell particularly good. We took this
idea and improved the diversity of microbial aroma production. Neroli was a highly rated scent in the study, and
we decided to add this fragrance to our system.
In addition, neroli alcohol is an intermediate in the synthesis of other important spices. At present,
neroli alcohol is mainly synthesized by chemical methods, but the current chemical synthesis method is
not only expensive, but also causes environmental pollution. Therefore, it is of economic and ecological
value to construct a strain that can synthesize neroli alcohol. At the same time, it also laid a
foundation for our future realization of microbial production of various spices.
Interview
Jinrong Xu
the cadre of Taijiang District Health Bureau, Fuzhou City, Fujian Province
When our team went to the kindergarten affiliated with Fujian Agriculture and Forestry University for
synthetic biology education and popular science activities, we were lucky to meet Xu Jinrong, a cadre of
Taijiang District Health Bureau, Fuzhou City, Fujian Province. We introduced our project to him and
mentioned the microbial fragrance-producing night lamp we hope to make.
Xu inquired about the positioning and function of our products. After we answered, his suggestion is
that we need to clarify the product positioning and pay attention to the impact of the product on the
the human body, especially in the part of human depression.
Q: At what counter do you sell your products ?
A: our products are sold in the perfume area.
Q: What kind of people are your products suitable for ?
A: Adults can use it normally, and minors can use it under the supervision of adults.
Q: Does your product harm the human body?
A: The aroma substances produced by microorganisms are food additives approved in the national standard.
We strictly control the production efficiency of microorganisms and low-concentration aroma substances
will not cause harm to the human body.
Q: What are the raw materials of your products ?
A: Microorganisms and media
Q: How to quantify the production of your products ?
A: We mainly carry out production in the factory. At present, we only carry out production in the laboratory
without mass production. We have designed the model diagram of the product and hope to put it into actual
production after the test is completed in the laboratory.
Q: What factory are you docking with ?
A: One is a factory that produces beverages, and the other is a factory that produces night lights.
Summary: We need to think about how our project will be produced and sold. In addition, we need to
understand some laws and regulations and related process standards.
Zhejiang University of Technology -- Jie Sun
1.Graduated from Beijing University of Chemical Technology majoring in Biochemical engineering.
yeast metabolic engineering and genetic engineering.
2.Biosynthesis of terpenes, Journal of Bioengineering
Cofactor engineering and metabolomics analysis of the synthesis of tert-alcohol saccharomyces neroli.
Zhejiang Natural Science Foundation.
3.Global transcriptional regulation of Pantothenic acid synthesis in Saccharomyces cerevisiae and its
metabolome analysis.
Q： What are the advantages of using yeast as a chassis organism ?
A：Because yeast is safer, Genetic manipulation is convenient, Suitable as the terpenoid hosts. There are many
kinds of yeast. And yeast doesn't contaminate phages.
Q：What is the impact on the growth of yeast itself after yeast is modified by the metabolic flow ?
A: Different changes to yeast have different effects. Case by case. It is also possible to divide growth
and product accumulation into two separate stages by means of which they do not affect each other.
Q：What are the steps required to construct a flavor-producing strain ?
A：Eukaryotes such as yeast already have an endogenous MVA pathway. The general idea is to overexpress a
series of competing enzyme genes. Isoprene pyrophosphate (IPP) can be first enriched by yeast. Then the
plant synthase gene is inserted to produce the aroma
Q：The modified yeast is nutritionally deficient. How do we incorporate it into our system ?
A：The modified yeast cannot produce uracil, leucine, histidine on its own. The solution is as simple as adding
these components to the final culture medium.
Summary: After the interview, we realized the superiority of terpenoids produced by eukaryotes.
We want
to use E. coli and yeast for co-cultivation to give full play to the advantages of prokaryotes
(technologically mature) and eukaryotes (high potential) to build our light-controlled microbial aroma
production system.
A foreign teacher interview
In order to learn more professional oral English expressions and points for attention in defense answers, we came to the International College of FAFU to visit the foreign oral English teacher from Nigeria on October 12th. We gave him a brief introduction to synthetic biology and iGEM and told him about our project. He told us that expressing confidence is one of the most important things. We need to have enough understanding and confidence about our project and improve our understanding of synthetic biology, so as to better overcome our nervousness. Defense speed is also important, to emphasize the key, layer upon layer in-depth. Through this conversation, we learned a lot, but also let us after the defense more confident.
Government Talent office interview
On October 11, 2021, our FAFU-China iGEM team arrived at the Talent Office of the Fuzhou District
Government
to further explore the development of team achievements.
First of all, we visited Mr. Wu Rihui, the head of the Talent Department, and asked our team what
good
direction and policy we have if we want to continue to develop our project. After listening
carefully,
we learned that funding and recognition are essential if you want to start a business from scratch.
And
it can be generally to solve two directions, one is through the game, such as planting farming, the
RongCheng gen youth entrepreneurship programs such as earning place, for the feasibility of the
project,
credibility basis and backing, the second is the solution for funds generally can get number from
youth
entrepreneurship to borrow a lot of, or access to funds from the corporate investment.
Later, we went to the hot Spring Park government Entrepreneurship Service Center and visited Ms.
Chen
Ling, deputy Secretary-general of Fuzhou Youth Innovation, to learn about the ways to start a
business
with a team project. I mainly learned that projects can be slowly implemented in the form of
competitions through some national competitions such as creative competitions, and I can learn and
visit
seniors who have participated in these competitions in the past to gain relevant experience.
In this activity, we learned some ways and policies related to entrepreneurship. It lays a
foundation
for the further development and landing of follow-up projects.
Commodity fair
Online Meetup
On May 29-30, 2021, our team participated in the first regional exchange meeting of this year, where we
shared and displayed our project topics for this year, and at the same time communicated to the teams
the preparation process of the team competition.
This exchange meeting (2021 China iGEM Online Meetup) is co-sponsored by Jiangnan University
Jiangnan_China, Shenzhen University SZU-China, Jilin University Jilin_China, and Lanzhou University
LZU-China (listed in no particular order) and guided by CCiC. A total of 42 teams were present, of which
28 participated in the project display.
When the project sharing was over, we ushered in a speech by Chen Jionglin, Secretary General of the
CCiC Executive Committee. He introduced the organization profile of CCiC and its development history,
its status and role in iGEM, and what kind of support and help each team can get from CCiC. Finally, he
also announced the 8th CCiC Conference to be held at Fudan University this year.
Of course, fortunately, after the meeting, we successfully met with SZPT-CHINA. After understanding the
content of the two parties' projects, our two teams reached an initial willingness to cooperate. This
facilitates the follow-up work of the teams on both sides.
CCiC8
CCiC is the abbreviation of the China iGEM Community Conference. This year's 8th CCiC was launched online and
offline due to the impact of the new crown epidemic. From August 28th to 29th, 79 teams shared their research
results and experience through team presentations and poster presentations at CCiC's online and offline venues.
During the meeting, we actively maintained communication with other teams. While making suggestions, we also
gained many valuable opinions.
After the project presentation, the judges put forward helpful suggestions on our presentation methods
(for example, we need to focus on the use of high-value-added products in the follow-up), and also affirmed the
effectiveness of our report.
After participating in CCIC, many teams approached us to solve different problems through cooperation with us.
The SZ-SHD team approached us with the idea of using our fragrance-producing strain to solve the deodorization
problem of their hair removal cream. The team from Tongji University also came to us at that time and wanted to
work with us to solve the environmental governance problem. They face the problem of sulphide odor in treating
sewage. Our linalool reacts with sulfur compounds to eliminate the odor effects of sulfide.
In future work, we also want to use our fragrance-producing strains for more activities that are
beneficial to humans, rather than just building different fragrance-producing strains. We hope to
develop some applications, so that more people can understand fragrance, love fragrance; So that people
in the process of use, its physiological and psychological level can really benefit.
Social practice
We distributed perfumes related to linalool and neroli (formal commodities) on campus and invited students to fill in the questionnaire or scan the TWO-DIMENSIONAL code on the perfume to write and fill in the questionnaire. (The photo has been taken with the consent of the parties concerned)
A total of 180 paper questionnaires were collected on campus and in nearby communities, of which 84.38%
said they would use products containing fragrance. Fresh and refreshing scents were the most popular,
followed by floral and fruity scents, with jasmine and mint topping the list. To our surprise, 80.77% of the
participants were willing to accept the use of microorganisms as the carrier of aroma production.
The main reasons for not accepting microbial fermentation for aroma production were fear of microbial leakage
and fear of changes in aroma itself. About 80% of people use products containing fragrances to regulate their
mood and help them sleep. They also hope that we can apply the project to perfumes,
aromatherapy, and other daily articles.
We have adopted the suggestions of the masses, hoping to design a small night light for microbial
fragrance production, so that our project really approached people's lives. At the same time, we also
consider the diversity of microbial fragrance production, let the microbial fragrance of jasmine and
mint is our future direction of efforts. We have also fully considered people's safety concerns about
microbial fragrance production. We will build double barriers from the hardware and the strain itself to
ensure the safety of our projects and products.
Visit factories
Under the guidance of the instructor, we visited a teaching factory that produces drinks. The person in charge
of the factory introduced the technological process of juice processing in detail for us. The functions of
agitation ultrafiltration equipment, high-temperature instant sterilization equipment,
trinity canning equipment and other equipment are very suitable for making a premix fermentation tank designed
by us.
We explained the idea of making a premix fermentation tank to the person in charge and the person in
charge approved our idea, indicating that the production line can indeed be used to produce sterilized
liquid culture medium, and mix the powder with the culture tank in the trinity canning equipment.
At the same time, the person in charge suggested to us that, for example, the fungus powder should be
wrapped in special materials or devices so that the canning equipment can mix the quantity of fungus
powder with the fermentation tank and then seal it.
In order to meet the factory's production requirements and users' requirements, we wanted to design a
special fermenter, which not only meets the factory's production requirements, transportation
requirements, but also enables users to use the fermenter directly without aseptic operation.
Summary
Silver
We did a lot of background checks before we decided on the project. Natural spices yield low, can not meet the
market demand, and the production cost is high. Nowadays, many spices are artificially synthesized by chemical
method, but the chemical industry will cause serious pollution to the environment. We hope to use microorganisms
to produce aromatic substances and realize their industrialization, in the case of meeting market demand, the
development of environmentally friendly industry. After the establishment of the project, we distributed
questionnaires and conducted interviews in the campus and community. The results show that most people recognize
the use of microbial production of aromatic substances, but also look forward to our research and development of
microbial incense production small night light. Safety and practicality were at the heart of the project, and we
interviewed many experts and listened to their opinions.
In terms of safety, one is how to avoid biological leakage, and the other is how to ensure that our volatile
products are safe. Our solution is to use nutrition-deficient microorganisms and supporting related devices, so
that microorganisms can not leave the culture medium; Even if microbes can leave the medium, they cannot
survive. Linalool and neroli produced by us are food additives approved by the state. They are very safe in
normal concentration and have certain pharmacological effects, which can relieve people's negative emotions.
After the biological transformation of chassis is completed, we will use gas chromatography to measure the
concentration of aromatic substances produced by microorganisms, and then ensure its safety before it is used in
the production of microbial aroma night-light.
In terms of practicality, we want to go in two directions. One side is for the factory, high density
cultivation, to mass production of spices; One side is for the family, low density culture, the production of
microbial fragrance small night lamp.
Last but not least, we did all the HP work legally and ethically. HP's results guide the development of our
project.
Gold
Our human practices have always influenced the design and execution of our projects.
In the early stage, we asked Teacher Artemisia argyi about the source of plant fragrance, namely volatile
organic acids and terpenoids, in the form of interview. We followed the MVA pathway to find all terpenes
(ionone, Citral, Citronellal, Nerol and Linalool) in the downstream, and consulted Professor Tian Yuting about
the use of aroma substances and matters needing attention. For safety reasons, Tian asked us to use the flavor
additives approved in GB 2760-2014 (National Standard for the Use of Food Additives). By referring to GB
2760-2014, we have greatly narrowed the range of target products. Finally, according to teacher Zhang Weiyin's
suggestion, we chose linalool and neroli, which have mild smell and the lowest synthesis difficulty, as the
products of chassis biology.
Neroli and linalool are mild in taste and easy to produce, making them suitable for early exploration in the
project. We then conducted a questionnaire on smell preference. The statistical results show that the public has
a high recognition of the taste of linalool and neroli alcohol, and also has a recognition attitude towards the
microbial production of fragrance and the production of microbial aroma night-light. On this basis, we began to
design the hardware of microbial aroma night-light.
With the pandemic, we don't have time to reinvent chassis biology from scratch. So we got in touch with Sun Jie
from Zhejiang University of Technology for help. Teacher Sun Jie recognized our project very much and gave us
saccharomyces cerevisiae strains that could enrich IPP, which greatly accelerated our experiment process. In
terms of biosafety, Teacher Sun Jie told us that the saccharomyces cerevisiae we used was Leucine, Histidine,
Uracil, which could only grow in a specific medium and would not survive without the medium. This inspired us,
and we also hope to engineer our chassis biology into a composite nutrient deficiency strain.
On the other hand, we got in touch with the factory and told them about our device idea and asked for their
opinion. Combining various opinions, we finally designed a small night light and its accompanying ferment tank
that not only meets the production needs of the factory, but also is convenient for transportation and buyers to
use.
There are many trade-offs to consider when it comes to the productivity of aromatic substances. If the
production efficiency is high, it may produce high concentration of volatile gas, and we are concerned about its
adverse effects on the environment and biology. For safety reasons, we prefer to screen strains with moderate
production efficiency for low-density culture. On the other hand, the light control element we used is directly
induced by blue light, but blue light has a negative effect on human body at night, so we can only use white
light source of about 15 lux (a distance of about 20cm) for induction (the induction effect is not as good as
pure blue light, and the light induction time is longer). In order to prevent blue light from the outside and
inside of the night lamp from passing through the lampshade, we plan to use a two-way blue light filter to block
blue light.
Finally, we also consulted relevant government departments on the follow-up development of the project to obtain
some technical, financial, legal and other assistance. We hope that after the project is mature, the project
will be promoted under the premise of ensuring safety.
Close the loop
Reference
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