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For strong attitude, high influence, and high impact stakeholders, we aim to constantly communicate with them to acquire their feedback. We will prioritize their demands and constantly revise our project based on their suggestions. Stakeholders classified
into this category are the most targeted customers and the business partners who share the benefits with us.
We classified individual flower lovers and floral design companies
who are the respectively the most and second prioritized target customers into this block. They are in huge demand for an acaricide like Dienamite, therefore they exhibit a positive attitude towards our product and
have the highest potential to interfere with our project development. Dienamite also have the highest potential to transform their lifestyle. Besides, potential business partners who shares the same interests with us
are also categorized into this block.
For strong attitude, high influence, and low impact stakeholders, we aim to constantly communicate with them while only selectively satisfying their requirements. Stakeholders endowed into this category are the target customers that are less prioritized.
We
classified large-scale farms, small-scale farms, forestry bureaus, and pesticide registration bureaus into this category. The former three are target customers that already have several acaricide options and considers
most about price, therefore Dienamite establishes fewer positive impacts on them. However, once they decide to purchase our product, they have the potential to influence our sales volume significantly due to their enormous
scale. For the registration bureau, the certificate they approved will directly determine the implementation of our product, but the success of our product will not establish significant impacts on them.
For strong attitude, low influence, and low impact stakeholders, our goal is to keep them informed. Stakeholders classified under this category show positive attitudes towards the emergence of our product but are not identified as our target customers.
We
categorized specialists from Center of Disease Control into this category. These specialists acknowledge the adverse effects of chemical pesticides, thus encouraging us to develop a syn-bio acaricide that is safe for
humans and the environment. However, they don’t normally encounter spider mites and are not our target customers.
For weak attitude, high influence, and high impact stakeholders, our goal is to regularly monitor their development and alter our business strategy in accordance to theirs.
We categorized competitors—mainly involving
other companies producing pesticides—into this block. Though they have a relatively negative attitude towards the development of our project, they possess a huge possibility to interfere with the development of our
project. While maintaining competitive relationship, we also learn from them by evaluating their business strategies.
For weak attitude, high influence, and low impact stakeholders, we aim to constantly inquire them to seek professional advice and consequently adjust our product design. Stakeholders endowed in this category are mainly specialists in different fields
whose advice is constructive to our decision making.
We classified syn-bio specialists, business experts, and bioassay specialists into this category. Although they express
low level of interests toward Dienamite since they are not our target customers, their insights are extremely significant for the ongoing development of our project.
For weak attitude, low influence, and low impact stakeholders, our goal is to regularly advertise our product. Although currently they might not be interested in our product, they have the potentials to become our target customer.
We
classified general public, especially those who are not frequently exposed to pesticides in their daily lives, into this block. They exhibit low attitudes at present but can potentially convert into any of our target
customer groups.
This section illustrates the ongoing motivation that drives the continuous execution of human practice activities. A consecutive mindset is demonstrated here, as the majority of the inspirations are inspired by the reflection of previous activities.
During the interaction with the stakeholders, our aim is to ensure that clear and effective two-way communication is executed. By preparing ourselves with concise interview guidelines and precise project description, the stakeholders can fully understand the purpose of our project and thus be able to articulate their concerns and suggestions effectively.
During the investigation process, we carefully analyze the information gathered from the stakeholders using a critical mindset. Their feedback is addressed and refined before practical implementation to best fit the progression of our project. Aside from that, we attempt to discover social inadequacies by evaluating the stakeholders' response.
This section highlights our efforts to pragmatically improve our project design. We incorporate suggestions and transform them into actions, continuously refining the feasibility of Dienamite to ensure it is both effective and responsible for the world.
This section conveys our dedication towards resolving real world challenges in response to social expectations. Once we identify any underlying inadequacies related to our project development, we oriented Education, Inclusivity, and SDG activities to hopefully improve the status quo.
Inspired by our research of spider mites, we embarked on our journey by first attempting to confirm the ubiquity of spider mites and figuring out the issues related to the current solutions to consolidate the necessity of our project. In order to collect more data and diverse perspectives from various groups of plant lovers, we decided to distribute questionnaires to a large crowd and obtain first-hand information that is convincing and factual.
Multiple questions were included in the questionnaire, aiming to obtain crucial information related to the ubiquity of spider mites, current strategies for eliminating them, and the corresponding user experiences. As shown in figure 3 and 4, from the 324 answers we acquired, approximately 85% were from individual flower lovers. Among them, more than 65.1% have experience of either encountering or being troubled by spider mites. Meanwhile, 54.8% of the participants resolved the problem of spider mites by purchasing chemical pesticides. The rest is still suffering from the inadequacy of traditional treatments (such as chili water) or directly but wastefully discarding the plants.
As implied, a high percentage of the individual flower lovers who participated in our survey have been bothered by the presence of spider mites. We can therefore confirm the significance and ubiquity of these obnoxious creatures.
We
also concluded from the survey that chemical pesticide is currently the most popular strategy used to combat red spider mites, despite mostly unpleasant user experiences. This suggests the limitation of available solutions,
as the most used strategy still exhibits various problems. We therefore believe that these conventional approaches are insufficient for addressing the problems caused by spider mites. In response, a new solution that evades
these inefficiencies would be in great demand. Despite this, from the results obtained, we discovered that the public’s awareness and familiarity regarding Synthetic Biology are relatively inadequate. Promotion and educational
activities for synthetic biology therefore came to our mind.
After realizing that a safe acaricide combatting spider mites is in demand, we started literature research to seek for an environmentally friendly substance that establishes exterminating effects on mites. We also wish to accomplish the expectations for egg eradication mentioned by the participants. Fortunately, we soon discovered the desired substance—7-Epizingiberene—a sesquiterpene that is safe to mammals, ginger-scented, and has fecundity-reducing and eliminating effects on adult mites.
After identifying the society’s lack of education towards synthetic biology, we attempted to conduct several activities to advocate it, aiming to simultaneously promote the concepts of our product and ignite others’ passion towards
this charming field. An Wechat official account called "SCIE iGEM" was established, which propagates simple syn-bio knowledges and advertises our product towards diverse age groups in a convenient and accessible way.
To address the negative feedback regarding the toxicity of chemical pesticides that is heavily mentioned in the questionnaire, we decided to investigate the health impacts they establish in detail. We therefore contacted Mr. Qin from Shenzhen Centers for Disease Control to gain more insights into it, aiming to further evaluate the necessity of our project.
Mr. Qin outlined numerous detrimental effects of chemical pesticides on human health, including skin disease, central nervous system damage, kidney dysfunctions, etc. Specifically in terms of acaricide, he noted, typical toxic chemicals such as dicofol, benzene hexachloride, and phosphamidon are involved, inducing diseases such as diarrhea and cardiovascular disorder. In response, we introduced our project Dienamite, a biologically synthesized acaricide with high target specificity and security. Mr. Qin, in return, enthusiastically acknowledged its potential and remarked that Dienamite might bolster the progression of sustainable development goals, especially the goal of ‘good health and wellbeing’.
Overall, Mr. Qin’s comments on chemically synthesized pesticides further consolidate the importance of our project, encouraging us to delve into it more. Besides, his mention of SDGs grabbed our attention. After researching, we
discovered that Dienamite has the potential to contribute to multiple of them, including Goal 2, 3, 9, and 12, namely Zero Hunger, Good Health and Well-being, Industry Innovation and Infrastructure, and Responsible Consumption
and Production. Commited to making the world a better place, the idea of promoting and implementign them thereby came to our mind.
Motivated, we started our journey to advocate global sustainable developments With a group meeting organized, we discussed our potentials and identified ways to broaden our coverage over various goals. Besides, through the discussion with Mr. Qin, we identified the highlights of Dienamite as being responsible for human wellbeing and environmental conservation. We hence decided to focus on advertising and marketing Dienamite using these qualities, engaging more target customers into considering our product.
Realizing the public's lack of awareness towards the impacts of chemical pesticides, we published an informative article on our Wechat Official account, educating people about their detrimental effects. Additionally, we also included
instruction teaching people to identify toxins involved in them, aiming to minimize the health influence these acaricides might cast on humans.
Since our ultimate goal is to successfully implement Dienamite to the market, our target customers should be identified and categorized into successive priorities according to their power of influence. By analyzing the negative impacts brought by the invasion of spider mites and the identities of the questionairre's participants, we classified two separate groups of people — the horticulturists and the farm owners — who are most susceptible to these destructions and thus might be willing to purchase our product. At this stage, we first focused on exploring the needs of large-scale farms, which requires massive amounts of pesticides combatting spider mites for better yields. We first contacted the owners of a strawberry farm that is constantly disturbed by the presence of spider mites.
During the interview, the owners suggested that spider mites emerge frequently throughout the year, causing severe destruction to their strawberries. ‘Sometimes not a single grain was reaped’, they described, these minuscule creatures often cause economic devastation to the farm. Regarding the solutions, the interviewees told us that they purchase chemical pesticides in the form of sprays from local retail stores to control the mites. They noted that the application of sprays requires massive amounts of labor work and time since appliance on individual plants is required, encouraging us to design automatic spraying facilities so that they could save costs from a long-term perspective. Additionally, they also underscored that although most chemical pesticides exhibit some efficacy, they establish detrimental effects on the environment.
The interviewees showed some interests towards our pesticide regarding its eco-friendly properties. However, they conveyed concerns regarding the price of Dienamite after we introduced the concepts of synthetic biology. Additionally,
they confided that they never wear or use any protection while spraying chemical pesticides and were genuinely neglectful of the health damage imposed by these pesticides. A general lack of interest was expressed when we emphasized
that Dienamite is safe for humans and pets. We therefore classified them into the fourth category of the user analysis pyramid, which consists of end users that have less demand for our product.
Although they were classified into the last category of the pyramid, some of their dissatisfactions with the current arrays of strategies inspired us to enrich our project according to their requirements. We took their complaints about chemical pesticides’ inadequacy to preserve the environment into account, which englightened us to enhance our project design by adding new components that evades this problem while also serving strong pest eradication capacity. We then started literature research and successfully discovered mite venom peptides(MVP), which derived from the predator of spider mites, thus exhibiting strong exterminating effects. Mostly significantly, these venom peptides are biodegradable, remaining only slight amount of residues after appliance and preserving water sanitation. Aside from that, the owners' commercial sources of chemical pesticides provided some guidance for our entrepreneurship activities and product design. Deeply inspired, we also designed our initial product in the form of spray. Meanwhile, since many farm owners like them only trust and purchase products from local retail stores, we started to realize the importance of business collaboration to increase the credibility and sales volume of our product. Additionally, the idea of designing automated facilities was also elucidated from the interview.
We were extremely astonished when the interviewees told us that they never perform any forms of protection, not even masks while using chemical pesticides. Only vaguely acknowledged that most chemical pesticides contain poisonous
substances, they were completely unaware of the severe and adverse health effects these pesticides could evoke. We therefore realized the importance of educating farmers about the dire impacts of chemical pesticides and advocating
the usage of protective measures when applying these products. With these aims, we conducted an educational activity with farm owners, hoping to enhance their awareness of self-protection.
Realizing that results from merely a single strawberry farm might not be sufficient to represent the perspectives of all the farm owners, we decided to conduct another interview with large-scale plantation owners. We thus interviewed Mr. Tao, the owner of a tea plantation located in Hangzhou, who possess a farm that cultivates different crops, operates more modernly, and is located at a different terrain in contrast with the strawberry farm.
Regarding the presence of spider mites, Mr. Tao also told us that his farm has long been troubled by them. After the infestation of spider mites, the quality and appearance of the tea are significantly affected as the leaves turn
yellow and withered, which in turn reduces the selling price considerably.
By this stage, we had incorporated mite venom peptides (MVP) into our product design. After we introduced 7-Epizingiberene and MVP and their
corresponding functions, Mr. Tao showed huge interests and enthusiasm. He suggested that our product has high feasibility and is suitable for farms since MVP is degradable under UV light and 7-Epizingiberene is liposoluble.
These properties ensure that only minimal amount of residue remains on the crops, aligning closely to the regulations of the national Agriculture Ministry and making it extremely safe for human consumption. Despite his approval
in the overall project design, he did convey concerns for the idea of designing automated facilities. Since his plantation is located at a mountainous terrain, it might be extremely difficult to install equipment.
Overall, the interview with Mr. Tao promoted our project development on Entrepreneurship. His approval on MVP and 7-Epizingiberene made us realize that the liposoluble and degradable properties of these substances are the highlights
of our project, and we should focus on advertising these features when we promote our products to farms that plant crops for consumption purpose.
However, we believe that modern industrial farms like his cannot be classified
as our angel end user. Although Mr. Tao seems more intrigued compared to the owners of the strawberry farms, he still conveyed similar considerations on the cost of pesticides. Moreover, he frankly suggested that safety of
pesticide application wasn't his main concern when buying pesticides. Therefore, we opted to place modern profitable farms also into the fourth category of the user analysis pyramid but at a position that is higher than local
domestic farms due to their emphasis for pesticide degradability.
Inspired by his suggestions on the idea of automatic spraying equipment, we decided to customize several forms of facilities based on the terrains of the plantation (Inclusivity link). For farms on mountainous terrain, we designed a facility that has a similar shape to a backpack, therefore achieving the requirements for portability and high capacity simultaneously. Meanwhile, for farms located on plain areas, we designed automatic irrigation equipment as shown in figure 9, which eliminates the need for labor costs.
Having both major communities of agriculturist addressed, we forward our HP journey to the next group of potential spider mite victims, the horticulturists. Apart from the catastrophe two-spotted spider mites incurred in agricultural production, they also cause severe damage on horticulture since their invasion results in the shriveling and yellowing of leaves. Therefore, it could be concluded that the horticulturists are equally susceptible to the invasion of red spider mites and hold the potential of being our target customers. We aim to also explore and analyze their needs. The first step of our exploration was with Shenzhen Forestry Bureau, a government institute responsible for the planting and maintenance of urban greenways.
We mainly interviewed Mr. Lin, the technician of the Green Space Management Division of the Shenzhen Urban Management Bureau who formulates pest control policies and purchases pesticides. Meanwhile, two other workers from the bureau
that physically executes the policies and uses pesticides also participated in our interview.
Mr. Lin told us that spider mites are pervasive in Shenzhen and often invade well-liked plants including Rosa chinensis and Plumeria obtuse. Regarding current exterminating strategies, he suggests that chemical pesticides are the most
frequently used method. When we asked about the drawbacks of these pesticides, Mr. Lin said that chemical pesticides often possess foul smell, and the two workers also described symptoms of dizziness while applying the pesticides. As
we connoted Dienamite's safety for humans and pets, they were extremely intrigued. Additionally, Mr. Lin also underscored that spider mites have a short life cycle yet overwhelming reproductive ability, resulting in a drastic speed of
resistance development. Thus, a specific chemical pesticide usually cannot be persistently used for over two years: different brands of pesticides are often used concurrently to prevent the development of resistance.
Humbly, we learnt that different pesticides are applied at according time periods to eliminate eggs, larvaes, and adults depending on insect growth cycles. Thus, the classification of stages will make the elimination more thorough and
decrease the chance of future infestation. However, Mr. Lin pointed out how most insects are regional, meaning their growth cycles are distinct at different latitudes. Hence, pest control policies often vary depending on landscapes and
regions..
Given the workers' and Mr. Lin's concerns about pesticide smell and toxicity, we believe they seek a safer option. Dienamite, which is highly effective and less harmful, serves as a suitable substitute. More importantly, greenways are a common thoroughfare, so a nontoxic product is essential to ensure safety of pedestrians. Hence, we assume that forestry bureau has a higher demand for products like ours compared to the agriculturists we interviewed. Subsequently, we placed them at a position higher than the previous two farms in the end user analysis pyramid and prioritized their requirements more. Nevertheless, we still believe that forestry bureaus aren’t our angel users due to their consideration on price with limited budgets.
Enlightened by Mr. Lin's emphasis on spider mites' exponential reproductive rates and chemical acaricides' incompetence in averting this challenge, we conducted research to seek for another substance combatting this problem. With
the presence of spider venom peptides discovered and incorporated, multiple ion channels of the spider mites can be targeted, thus crucially reducing the possibility of resistance development.
Moreover, Mr. Lin’s classification
of egg, larvae,and adult mite elimination inspired us to characterize our products into corresponding stages. For the pre-infestation stage in which only eggs are present, 7-epiZingiberene and 9-Hydroxyl, 10,11-epoxy zingiberene
can be used to establish fecundity-reducing effects. Meanwhile, if the plant is already infected by adult mites, 7-Epizingiberene, 9-hydroxy-10, 11-epoxy zingiberene, spider and mite venom peptides can be used concurrently
to eradicate them.
Besides, the information of the regional variations in spider mite lifecycles also drove us to improve our product design. To make our product more inclusive to broader users, we devised a topographic
map that includes different life cycles of spider mites in different regions. It also contains information about the suitable time for using prevention techniques and exterminating techniques respectively.
Previously, our interview with Mr. Lin prompted our understanding of the importance of safety when cultivations face large public. Thus, it came to our mind that horticulturists for public service industries - such as hotel or restaurants - might also hold similar concerns when purchasing pesticides. Furthermore, cultivations placed in public for asthetics are naturally victims of spider mites as undesirable infestation sceneries ruins their appearance. Therefore, intended to gain a deeper understanding, we conducted an interview with Ms. Yen, who is a professional horticulturist currently running a flower arrangement studio, designing and selling flowers mainly to hotels and offices.
Similar to other interviews we conducted with forestry bureau, Ms. Yen declared that red spider mites can detrimentally affect the appearance of plants and may lead to decline or death. Once these situations happen, Ms. Yen and
her company will carry the responsibility of exterminating the mites while replacing damaged plants with new thriving ones. These measures will cause excessive financial losses and unnecessary burden to Ms. Yen’s company.
When inquiring the current solutions she applies to eliminate spider mites, we realized she used traditional methods such as spraying chili water and chemical pesticides simultaneously. However, in her scenario, both solutions
contain drawbacks—chemical pesticides have foul smell and toxic ingredients; chili water causes sneezing of the passersby. As Ms. Yen suggested, these situations will arouse the unsatisfaction of the customers who are constantly
in contact with plants. Besides, she also mentioned her need for an irrigating facility that can be placed individually on each plant. With the help of that, she said, the need for constantly checking and applying pesticides on
plants at different spots could be eliminated.
As indicated by the interview, most horticultures from floral design companies are placed in hotels and offices located around human, highlighting the need for safe and odorless pesticides. Since Dienamite only exhibits a slight scent of ginger and is safe to humans and pets, its features align quite closely to the floral design companies’ requirements. Besides, as conveyed by Ms. Yen, these types of companies also have relatively high acceptance for price. Consequently, we decided to treat them as one of the major target customer groups.
Ms. Yen’s comment on irrigation facility drove us to enrich our current equipment designs. Being inspired, we designed a new equipment that can be used at prevention stage, during which 7-epiZ and 9H10epoZ are automatically dripped onto the plants and establish repelling and fecundity-reducing effects on spider mites.
Words of Ms. Yen about how pesticides applied near people require some of the key characteristics of Dienamite - safe, odorless, and effective - provided us with new insights regarding potential target users of Dienamite. Given that current arrays of solutions pose certain threats to human health, we consider people who cultivate plants at home potentially holding urgent need for Dienamite. Therefore, we settled on interviewing Ms. Ada, an influencer we found on Red, who is an individual florist and has a small garden in her home. From her posts, we discovered that she has more than ten years of flower growing experiences and has been disturbed regularly by spider mites.
During the interview, Ms. Ada emphasized how spider mites behaved as a major threat to her garden. These malicious creatures bothered her every year and brought huge destruction to her beloved plants. Once, she had been forced to
discard her favorite vine plants that her mother gave her due to the infestation of red spider mites. The vine meant a lot to her, and she was immersed in huge sorrow and frustration when the plant lost its vitality. As a solution,
she currently uses chemical pesticides. She then conveyed her huge disatisfaction towards them, comdemning the foul smell of chemical pesticides that affected her family’s living quality. Besides, Ms. Ada was troubled by potential
adverse impacts on her and her family’s physical health, especially with the presence of kids and pets. Accordingly, she expressed huge enthusiasm and anticipation towards our product when we informed her that Dienamite is completely
safe to humans and pets. In addition, Ms. Ada also shared her struggle of losing her plants even after a thorough elimination of the mites. Her hopes are pinned on our products.
Introducing the synthetic process foundation of our product, we informed her about the possible high cost due to complex production method. Surprisingly, she demonstrated huge tolerance to the price of pesticide. Her acceptance ranges
below the price of plants.
Based on her feedback, we set our eyes on the target customers analysis again. From Ms. Ada’s enthusiastic reaction towards the eco-friendly and safe properties exhibited by Dienamite, we concluded that amateur florists are in need of a safe syn-bio produced pesticide. Due to the fact that they spray pesticides at home, the solution may diffuse and linger around the interior for a relatively long period. If toxins are inhaled, people will be severely affected. Compared to farm owners and forestry bureaus who have less exposure to toxic pesticides, amateur florists prioritize safety the most and obtain greatest demand for a secure and reliable biopesticide. Furthermore, reside duration at home exceeds public areas, such as hotels and restaurants, their urge for safety would reasonably be stronger than that of floral designers for service industries. Besides, amateur florists' tolerance of the higher price aligns with our product’s monentary properties. Hence, possessing greatest demand and highest tolerance, amateur florists are classified as the angel users of our product and are placed at the top category of our end user analysis chart, higher than the hotel floral designer given their lower expectation of product cost. Their requirements will be considered and satisfied prior to other groups of end users.
Unexpectedly, Ms. Ada told us that mite infections will affect the immunity and cause death of plants even when pests are eliminated using chemical pesticides during recovery. We became highly aware of this problem and are determined to
seek a solution that combats this issue. After extensive literature research, we discovered Harpin—a glycine-rich protein that bolsters plant immunity and promote plant growth. Harpin perfectly resolved our concerns, propelling us to incorporate
it into our project and characterize a new stage known as post-infestation stage. In which, mite-infected plants recover with stronger immune defense.
Singular characters like Ms. Ada also enlightened the idea of individual differences
in terms of pesticide application. In particular, her anecdote about concerns for her family highlights the need for our focus on individuality in order to establish a complete outreach of our product, as this is not something simple generalization
could address. In spite of this, adjusting our product for every single individual users would not be feasible. After intense discussion, we concluded that we should instead reverse our mindset and acknowledge impediments to our product that may
hinder individual usage of pesticide. We subsequently refined our goal of inclusivity - to exterminate barricades of equal pesticide usage to the largest extent.
Through the interview with Ms. Ada, we also found a pervasive consciousness gap among amateur florists. The majority of them lack the awareness of pest preventation. Usually, no measures are taken to exterminate the eggs of spider mites, nor to repel them, which in turn results in massive invasion. Aiming to reduce the destruction caused by subsequent adult mites, we conducted an educational activity to increase the preventive awareness of amateur florists.
During our production of 9-hydroxy-10, 11-epoxy zingiberene in E. coli, we met difficulties associating with the construction of a plasmid. Ideally, after successful construction, the plasmid should contain five enzymes including NPPS, Mvan4662, ZIS, ZPO, and CPR in order to produce 9-hydroxy-10, 11-epoxy zingiberene. However, since the length of the complete plasmid will be approximately 13000bp and the sticky ends we used are only 3 bases long, the construction became extremely difficult. After failing for a month, we decided to seek help from specialists who expertise in E. coli plasmid construction.
We used Wechat to communicate with Mr. Jin, a PhD student from Fudan University and is experienced in plasmid construction.
We conveyed how we planned to insert two DNA fragments that are both approximately 2000bp into a
plasmid that has initial length of 9000 bp and he suggested that it was slightly difficult to insert short fragments into a long fragment. He then advised us to increase the proportion of short fragments and enzymes in the
Golden Gate Assembly system and augment the time duration for the reaction.
Mr. Jin's feedback about the difficulty of assembling short fragments onto one long fragment inspired us to reduce the number of long fragments. We decided to separate the long fragment of around 9000bp into four segments, expressing three enzymes and the original vector respectively. Thus, we obtained a Golden Gate Assembly system consisting of 6 shorter fragments compared to the initial design.
We utilized 6 fragments to assemble the plasmid. During the procedures, we followed Mr. Jin’s advice of increasing the ratio of short fragments and his advice of prolonging the reaction time. After a few weeks’ attempts, we successfully constructed the plasmid that is 13000bp in length (link to engineering success). The following figure reveals the correct sequence obtained without mutation.
Through Mr. Jin's advices, we have successfully overcame the challenge of constructing large plasmids. Specific experimental processes could be viewed in our Engineering Success webpage, Cycle 2-1 (link to engineering success) Our detailed experimental documentation serves as a valuable resource for future iGEM teams undertaking similar projects, thus establishing helpful impact to the large iGEM community.
When we were conducting experiments of the spider venom peptide branch, we also encountered multiple difficulties. At this stage, we have already expressed SVPs(spider venom peptides) engineered with Galanthus nivalis agglutinin (GNA) in the precipitation of E. coli. This heralds the fact that our peptides are not secreted successfully into the periplasm of E.coli, resulting in unsuccessful folding. In spite of our attempts on signal peptides, protein modifier SUMO, and secretion systems, this problem remained unsolved. Therefore, we decided to contact the authors of different literature research in order to seek professional support from them.
We successfully contacted and interviewed the prestigious Professor Glenn King from the institute for molecular bioscience in the University of Queensland, who’s the group leader of a team specialised in using spider venom peptides
to solve nervous system disorders and the publisher of the research papers "Insect-Active Toxins with Promiscuous Pharmacology from the African Theraphosid Spider Monocentropus balfouri" [1] and "Spider-Venom Peptides as Bioinsecticides"
[2].
We asked Professor Glenn King several questions related to the selection of signal peptides and secretion systems. He showed approval on our choice of a SUMO modifier initially, then suggested us to place our 6xHis
tag at the N-terminus and try MalE as a signal peptide. He encouraged us to use different combinations of signal peptides and SVPs to seek for the most suitable one for each type of SVP. Regarding signal peptides, Professor
King also pointed out that most venom peptides naturally carry sequences that code for a signal peptide, yet these signal peptides are insignificant as they won't contribute to secretion to the periplasm, nor would they enhance
toxicity by any means. As for secretion systems, Professor Glenn King commented that they are not efficient in secreting toxic peptides extracellularly as they themselves have attempted multiple times and failed, though the
secretion systems might have some positive effects on secreting proteins into the periplasm.
We decided to follow his suggestion of trying multiple signal peptides. Our experimental design incorporated MalE and PelB, testing various combinations, including newly constructed SVP-GNA plasmids engineered with or without SUMO
and coupled with either MalE or PelB. Following Professor King's mention of secretion system's impact on protein secretion into the periplasm, experiments with secretion systems were proceeded with the means of, not extracellular
secretion, but elimination of inclusion bodies. From the vast variety of secretion systems, we have chosen G1M5 as our initial attempt.
As for Professor King's insights on naturally occurring signal peptides within venom
peptides, it inspired us to undertake optimization efforts for our newly identified MVP sequence PpVP1F. Given the signal peptides pose no effect on the overall toxicity of mite venom peptides, we decided to truncate the corresponding
sequences, giving a modified version of the original MVP, PpVP2S.
With the incorporation of signal peptides, bands of protein expressed in the supernatant were still not observed. We quickly launched the second method of employing a secretion system, in our case G1M5, to resolve this as mentioned.
We engineered a representative of our venom peptides, rCtx-4, with G1M5 on vector pET28a, giving pET-G1M5-SUMO-rCtx-4-GNA. Aligning with Professor King's expectation, SDS-PAGE results reveal successful folding of venom peptides
intracellularly. This phased success is largely significant in spite of unsuccessful extracellular expression.
Moreover, the truncated version of MVP, PpVP2S, was revealed to have much greater lethality
than the original version PpVP1F, with 100% lethality of PpVP1S within 2 days and 83.64% lethality of PpVP2F after 72 hours. This marked a great achievement in our experiment.
Along with the guidance of Professor King, we have explored a variety of expression vectors, incorporating signal peptides, sumo tag, and ultimately G1M5, at the end achieving sucessful protein expression. Through our exploration, we have established a document with clear results corresponding to each attempt. This documentary hence have reference value in E.coli vector selection for expression of cysteine-rich peptides for the iGEM community.
Despite the increased expression of spider venom peptides in the supernatant, we still didn’t achieve secretory effects. Luckily, Professor Glenn King kindly introduced us to the renowned Professor Elaine Fitches from Durham University. Professor Elaine is the co-inventor of GNA and the author of "Spider venom neurotoxin based bioinsecticides: A novel bioactive for the control of the Asian citrus psyllid Diaphorina citri (Hemiptera)" [3] and "Enhancing the oral and topical insecticidal efficacy of a commercialized spider venom peptide biopesticide via fusion to the carrier snowdrop lectin (Galanthus nivalis agglutinin)". [4] With her help, we wish to enhance our experiment design on the venom peptide branches further.
Professor Elaine further specified the functions of GNA to us during the interview. She emphasized that GNA showed contact toxicity with pests such as aphids and is very likely to establish similar effects on spider mites. However, regarding the secretory effects, she commented that E. coli would not be an efficient chassis to achieve that. Indeed, she encouraged us to use P. pastoris as the chassis to obtain the secretory function and to increase the yield of the protein. She suggested that with the use of P. pastoris as the chassis, the production process would be scalable and industrial level production would be feasible. Besides, protein purification using nickel affinity chromatography, and the use of fermenter are also recommended to enhance the protein’s purity and yield respectively.
After intense discussion, we decided to attempt using P. pastoris as the chassis to eventually reach secretory effects. We modified our experiment procedures to suit a different chassis and optimized all codons of our venom peptides for P. Pastoris. In addition, we also listened to her advice on protein purification.
Following her suggestions, we are still working on the purification of venom peptides expressed via E.coli. We had employed purification through 6xHis tag at this point, though ideal results are yet to be achieved. We have
continued preceding procedures without purification and reached monumental success in venom peptides.
Still aiming for optimized venom peptide expression, we forward the experiment and switched expression chassis to
P. Pastoris . We have successfully inserted fragments expressing SVP-GNA into yeast. Though we haven’t reached the stage of expression due to time limitation, in October, we will try to verify the yeast using colony
PCR and we will ferment them to obtain protein secretion .
Results of P.pastoris excretion are yet to be achieved. We successfully obtained the yeast cultures. However, due to time limitations, we weren't able to perform colony PCR to verify our results. In the remaining time of October, we will continue exploring P.pastoris as the chassis, including the validation, fermentation, and expression of various designs. We would furthermore compare the secretion efficiency of both P.pastoris chassis and E.coli chassis to provide reference for futre iGEM teams.
After we successfully synthesized 7-epiZingiberene and 9-hydroxyl-10, 11-epoxy zingiberene and augmented the expression level of the spider and mite venom peptides, we decided to conduct bioassays on spider mites to verify the functions of them. However, we couldn’t obtain commercial sources of spider mites, and we didn’t have an accurate protocol for executing the in vivo experiments. Thus, we wish to seek help from a stakeholder that is authorized for performing bioassays on spider mites to ensure the accuracy and credibility of our results.
We conducted an interview with the prestigious Professor Huang from South China Agricultural University, who’s research concentrations are in natural source pesticides, pesticide toxicology, and pest control. He had conducted various
in vivo experiments of agricultural pests and is authorized by the Ministry of Agriculture and Rural Affairs.
During the interview, he conveyed the detailed procedures and precautions for conducting bioassays of spider
mites, including the appropriate temperature, humidity, and food source required.
Due to the difficulty of operating bioassays, we decided to cooperate with Mr. Huang. We biosynthesized the SVP and MVP and send the samples to him. He then assisted us in conducting spider mite bioassays that verifies the effectiveness of SVP and MVP in order to obtain results that are more accurate. Meanwhile, for the repellant experiment of 7-epiZingiberene, 9HZ, and 9H10epoZ, we decided to conduct bioassays on spider mites on our own based on the advice of Mr. Huang.
Following the instructions of Professor Huang, we had successfully conducted bioassays on spider mites with both venom peptides and the three sesquiterpenes. The results of the bioassay are shown in the following figure. Successful repellent effect was observed for sesquiterpenes and high lethality was concluded for all of our venom peptides. In particular, our newly discovered mite venom peptide PpVP2S exhibited a high 100% lethality in merely 2 days, with 19/20 female Tetranychidae urticae exterminated within 24 hours. The exceptional bioassay results for PpVP2S provided a strong foundation for our patent application, which we have successfully submitted.
We collected the information of the interview and produced an experiment protocol that includes the procedures for conducting bioassays on spider mites. The results of the bioassay provided strong support for our patent application.
With the initial project design refined after incorporating the suggestions of multiple target customers and specialists, the new design is ready for final evaluation. We interviewed Ms. Ada again, who’s classified as the angel user of Dienamite, to ensure our final design align with their demands. We also aim to seek for further adjustments.
We conducted an online interview with Ms. Ada to present our final design, and she expressed huge satisfaction with our work. She praised the inclusion of a regional map that outlines various user instructions, noting that it enhances the credibility and reliability of Dienamite while significantly improving user experience. Ms. Ada was also enthusiastic towards Dienamite’s staged protection (prevention stage, elimination stage, and ultimately protection stage harnessing 7-epiZingiberene and 9-Hydroxyl, 10,11-epoxy zingiberene, mite and spider venom peptides, and harpin protein respectively), which leads to more effective elimination. We shared the estimated price of our product, which she found to be perfectly acceptable. However, she raised concerns about the product's shelf life and suggested us to reduce the pesticide volume for more convenient storage.
The interview with Ms. Ada made us realize the importance of designing user-friendly facilities and instruction books. Considering amateur florists’ low frequency of use, we designed packages that is disposable and portable, with the capacity of each being low enough for one-time use. This fulfills their requirements for accessible storage.
The final equipment design for individual florists is shown below. With the application of dry powder, the packages exhibit high portability. Besides, the separate packs deliberately prevents cross spoilage of different substances, ensuring the
ease of use for the target customers.
Meanwhile, realizing the significance of designing a user-friendly interface and instruction book, we included a comprehensive product page on Wiki. Target customers can access detailed information regarding the various components and functionalities of Dienamite. A users’ guide is also written, ensuring efficient and simple operations.
Bearing the ultimate goal of implementing Dienamite into the market, we soon proceeded into the exploration of product commercialization. After assessing the feasibility and ensuring the fulfillment of the target customers’ demands,
registration certificate is required before putting the pesticide into production. We therefore decided to consult with Mr. Yuan from the Institute for the Control of Agrochemicals to inquire about essential information based on pesticide
registration.
After registration, the pesticide can enter the market through several avenues. The primary approach we mainly focused on is to establish a company and leverage its resources to facilitate market entry. Desired to
obtain more information about company establishment, we found Mr. Jiang, who is the manager of Yanhua Yongle Biotechnology Company that specialized in developing, large-scale producing, and marketing pesticide products. Given the alignment
of his expertise with our goals, we believe that valuable insights can be obtained from the interview.
Apart from pesticide registration and enterprise establishment, we also aim to identify potential collaborators who might contribute
to advertising and selling Dienamite in the future. Inspired by the previous interview with the owners of a small-scale strawberry farm, we thus reached out to Mr. Yu, the manager of GuangHui Agriculture Company, an enterprise that primarily
distributes pesticides to both horticulturists and agricultural farms.
Mr. Yuan briefly introduced the procedures for pesticide registration to us during the interview. Stunned, we discovered that excessive amounts of materials need to be submitted to multiple bureaus and numerous experiments need
to be conducted before the approval of the certificates. During the interview, Mr. Yuan asked us about the components of Dienamite. He told us that both the sesquiterpenes and venom peptides are novel substances that haven’t
been registered before. Hence, he strongly encouraged us to apply for patents to protect our discovery.
Meanwhile, during the interview with Mr, Jiang, we gathered essential information regarding the establishment and
management of an enterprise in its early, middle, and late phases. Export methods, exit strategy, and various channels for gaining financing revenues were mentioned. Additionally, Mr. Jiang emphasized that before exporting
the pesticides, they need to undergo multiple rounds of registration according to the policies of different countries, which prompted us to reflect on the complexities involved.
When we were communicating with Mr. Yu,
he shared his opinions regarding sales and advertising strategies. He told us that his company usually gather a throng of crowds and perform in vivo experiments on pests publicly using newly launched pesticides, strictly conforming
to the regional safety regulations. This assists with verifying the efficacy of the pesticides and bolster the brand’s credibility among farmers. We then introduced our product Dienamite. He unveiled great interests and conveyed strong
willingness to explore future partnerships, acknowledging that syn-bio produced acaricides possess bright prospects due to their significantly reduced resistance development rate.
The intricate processes for pesticide registration raised our concerns, prompting us to search the relevant policies online. We soon realized that the materials required are submitted through various platforms and examined by multiple
agencies. Most frustratingly, the policies detailing the information are spread across different websites, making it extremely challenging to figure out the proper sequence for submitting different documents.
On the
other hand, the interview with Mr. Jiang made us reflect on our current development plan. Indeed, his practical experience revealed the intricacy that we had not yet considered. Meanwhile, his emphasis on the variation in registration
and export policy inspired us to explore the strategies of other countries, enabling the global use of an environmentally friendly acaricide like Dienamite.
Additionally, the interview with Mr. Yu inspired us to explore
different modes of product promotion. Despite conducting public experiments on pests according to the governmental safety regulations for local advertisements, we also considered strategies of broader scopes, involving livestreaming,
broadcasting, etc.
Addressing the issues for protecting our invention, we listened to Mr. Yuan’s advice of applying for patent. After researching the process for patenting and consulting with authoritative patent agencies, we drafted and submitted
our patent application to the State Council, waiting for approval.
We further explored the business concepts and models that Mr. Jiang had mentioned and incorporated some of them into our development plan, including
the sections of revenue streams and exit strategies. Meanwhile, Mr. Yu’s advice on advertisements is also incorporated into our development plan.
Recognizing the inefficiency of the application process due to the lack of cohesive guidance for pesticide registration, we are determined to address the confusion by drafting a new proposal that integrates the all the governmental requirements which outlines the sequence of application. Harnessing this proposal, future iGEM teams and pesticide companies will be able to navigate the registration and production licensing process more smoothly and efficiently.
During the previous interview, Professor Glenn King mentioned his company Vestaron, which specializes in syn-bio produced pesticides. He emphasized the sustainable values at the core of his enterprise. As a prestigious example in this field, Vestaron inspires us to gain insights of sustainable agriculture and enhance our understanding towards the broader Sustainable Development Goals (SDGs). This motivated us to conduct a follow-up interview with Professor King to explore these core beliefs further.
‘Our mission is to harness biotechnology and our influence to convert more acres to sustainable pest management and enhance the sustainability value of agricultural land.’
Professor Glenn King conveyed Vestaron’s core values during the interview. He suggested, the significances of establishing an enterprise are not only about making profits, but also about leveraging the company’s resources
to contribute to conserving a greener earth. After we introduced our final project design, he acknowledged its sustainable concepts and encouraged us to promote the goal of 'Zero Hunger' while marketing Dienamite. Additionally,
he suggested that sustainability should be considered throughout the entire product lifecycle, including the packaging processes.
Deeply propelled, we firmly align with the prominence of SDGs. Hence, it was set as one of the core values of Dienamite. We also realized the necessity of educating the public about sustainable agriculture and horticulture. The idea of simultaneously advocating SDG and Dienamite to our target customers subsequently came to our mind after the interview.
With the identification of 'Zero Hunger' as one of our goals, we attempted to explore its correlation with Dienamite through extensive discussions. We discovered that the involvement of degradable venom peptides and liposoluble sesquiterpenes are highly compatible with the principles of food security, as minimal amounts of residues are remained. We deem, through our scientific approach to control mite populations, the adverse impacts they cause on agriculture can be minimized.
Through aligning Dienamite with the values of Zero Hunger, we advertise our products and advocate sustainable development goals with regards to that. We designed a poster and distributed it through several social media channels, engaging diverse groups of audience and hopefully raising their awareness on sustainable future.
Inspired by Professor Glenn King’s suggestion on sustainable packaging, we decided to investigate more about sustainable materials. After researching, we set our sights on biodegradable and recyclable plastics. Aiming to understand their difference, we reached out to Mr. Guo, the professor from the Chinese Academy of Environmental Sciences, who specializes in developing recyclable materials.
Similar to our previous discussion with Professor Glenn King, Professor Guo also acknowledged the prospects and sustainable natures of Dienamite during the interview. Meanwhile, regarding the differences of biodegradable and recyclable plastics, he suggested, recyclable plastic is more applicable as multiple companies have already implemented it. Thus, our need for recyclable replacement bottles confirms its high practicality. Besides, Professor Guo introduced the concepts of recycling metals to us, encouraging us to incorporate them into the design of our irrigation equipment and backpack equipment, thereby ensuring full consideration of sustainability in various types of facilities. In addition to their sustainable properties, being more affordable compared to unrecycled metals is another benefit mentioned by Professor Guo.
Following Mr. Guo's insightful recommendations on recyclable plastics, we decided to reach out to and collaborate with companies specializing in their production. Simultaneously, recognizing the benefits of incorporating recyclable metals, we plan to also explore partnerships with enterprises that focus on producing these materials. With these approaches established, we aim to strengthen our commitment to developing environmentally friendly packaging solutions.
Fortunately, we soon discovered an enterprise called the Green Recycling Plastics Company, which recycles both plastics and metals. After contacting the manager, Mrs. Qin, we visited the company and gained valuable insights into their recycling processes, which she refers to as 'responsible production'. She shared her company's approach to the 17 Sustainable Development Goals (SDGs) and we presented our project, Dienamite. Our progress in promoting these goals is evaluated professionally. Mrs. Qin expressed a strong interest in collaborating with us, particularly in collectively advancing SDG 12 (Industry, Innovation, and Infrastructure). Meanwhile, regarding the packaging materials, she recommended us using reusable polyethylene (PE), noting its environmental benefits and durability for transportation.
Thanks to Mrs Qin's participation in evaluating our product and providing valuable suggestions, we plan to implement reusable polyethylene as the main material for our product packaging. This expands on our idea of reusable plastic containers for Diena-spray. Three detachable bottles combined with reusable plastic maximizes our contribution to SDG. Moreover, individual packages (Diena-package) also benefit from this deliberate use of sustainable material as disposable wrappers no longer pressures burden on our fragile mother nature. Our passionate desire to align with SDG can not be fully described with only words, but our motivation will surely be amplified by all those affected and inspired by us.
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