Team:Ecuador/Human Practices
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Linda Guamán has a Ph.D. from the University of São Paulo in Microbiology. She has experience in engineering prokaryotes using synthetic biology with tools such as CRISPR and metabolic engineering approaches.
Carlos Barba has a Ph.D. from the National Autonomous University of Mexico in Biochemistry and Molecular Biology, where he worked with signal transduction systems. Since then, he has worked in synthetic biology optimizing bacterial pathways with different approaches.
In december 2021 our project aimed to detect the fungus Foc R4T by constructing a CRISPR Cas9-based biosensor producing a fluorescent signal as a positive result. When we presented the idea to Linda and Carlos, they told us that the project could work well for IGEM, but it would be better if we also propose a solution instead of a simple detection device. They both suggested us to work with bacteria that are from the banana soil microbiota to produce molecules that can act against the pathogen. While searching on the banana soil microbiota, we found two bacteria, Bacillus subtilis, and Pseudomonas putida, which are normally used as biocontrol. At that time we set out to enhance the production of lipopeptides (molecules with antifungal activity) with synthetic biology.
Ediner Fuentes has a Master's degree in Microbiology and works at the Gorgas Memorial Institute for Health Studies, within the Department of Virology and Biotechnology. He has extensive knowledge in biotechnology regulation within the Latin American region.
Our project idea was focused on the production of lipopeptides in Bacillus subtilis, and Pseudomonas putida. Therefore, attention was paid to the legislation related to the release of GMOs, following the statutes established within the Cartagena Protocol. Ediner recommended implementing safety systems to avoid possible horizontal transfer events.
Otto has a degree in chemistry with a specialization in biochemistry and molecular biology. He is an entrepreneur of biotechnology projects oriented to the agricultural sector and has extensive experience in RNAi technology.
The idea of using lipopeptides to counteract Foc TR4 was discarded due to legal loopholes in Ecuadorian regulations. We came up with the idea of using RNAi technology, of which Otto had ample experience. He helped us with flow charts for the design of dsRNAs. In addition to some design tips such as: Analyze the interactome of the gene to be silenced, that the RNA is not complementary to mammalian or pollinator sequences and that the sequences on which the design is based must be from reliable sources.
Salome has a Master's degree in Entomology and Plant Pathology. She is currently a PhD student in Plant Pathology at Oklahoma State University. She has worked with RNAi technology to inhibit the pathogenic effects of the fungus Pyrenophora tritici-repentis on wheat plants.
The project was in the design of long dsRNAs targeting virulence and pathogenicity genes of Foc TR4. Salomé recommended using the E-RNAi Design of RNAi constructs program. This program allowed us to predict the efficiency and specificity of the RNAi constructs by choosing the best scoring RNAi constructs and those with the fewest number of loops. This program also reported the primers for the amplification of dsRNAs from FOC R1 DNA.
Carolina Elizondo is currently doing her master’s studies at Heidelberg University and participated several times in iGEM with the team of Tec-Monterrey. She is part of the After iGEM Ambassadors program.
At that time the project was based on dsRNA production in E coli based on an inducible optogenetic system. Carolina felt that the dsRNA production module has no direct relation to the project problem and doubts the stability of the dsRNA. Therefore, we decided to change the production system using the L4440 plasmid and the inclusion of sequences that improve dsRNA stability.
Antonio is an Agronomist Engineer and is responsible for the Musaceae Program at INIAP's Pichilingue Tropical Exp. Station.
When we presented the project to Antonio, he suggested that Lamp's device should not be directed to banana growers, but to the regulatory agency Agrocalidad, in order to have better control of the disease. And with respect to the design of dsRNAs, of virulence genes, he mentioned that these have areas where generally many mutations accumulate, so we would expect there to be a big difference between Foc R1 and Foc TR4. He recommended us perform alignments between the two races to see the degree of difference. Antonio also helped us to define the delivery system for the trunk injection of the dsRNA in the plant. And also how to apply the biopesticide according to Agrocalidad regulations
Freddy Magdama holds a Ph.D. with dual degrees in Plant Pathology and International Agriculture and Development from Pennsylvania State University. He is an expert in plant pathology, population biology, molecular diagnosis of plant pathogens.
Efren Santos is a Senior Lecturer at ESPOL and has experience in genetic transformation of Musa spp. by Agrobacterium tumefaciens, detection of Genetically Modified Organisms (GMO) in crops and food.
Within the project, both experts questioned the origin of the genes that will serve as the basis for the design of the dsRNAs and the mode of application of the dsRNAs in leaves. They recommended using other genes that are being tested with mRNAs such as Six and doing trials on seedlings and not on leaves, since it makes it easier to obtain photographs to demonstrate the effect of the fungus on seedlings.
Member of the Association of Small Banana Producers "El Guabo" and representative of Pacidel S.A., a company dedicated to the production and export of bananas.
Our initial idea consisted of bacterial production of siderophores and glucanases in the soil rhizosphere to combat Foc TR4. We also had another focus on early detection of Foc TR4 by means of a LAMP diagnostic device in the field. Jorge pointed out that banana diseases are complicated to treat for organic producers and the need for effective biopesticides. He also mentioned the possible conflict we could have with the phytosanitary control agency of Ecuador because they are the only entity authorized to perform the confirmatory diagnosis of Foc TR4.
Alexis Vela is a biosafety specialist in the Ministry of Environment with a diploma in Biotechnology, Industry, and Business.
At the time, our project took its final focus on the production of dsRNA through bacteria. However, we had doubts about Ecuador's regulations of bio inputs.
Alexis suggested us to review the regulations for the registration of bio inputs, however, he expressed that Ecuador has a legal gap regarding dsRNA-based pesticides. He also emphasized avoiding mentioning that the product is derived from a GMO as this will create impediments in its registration. The risk analysis should focus on the product and not on the technology that was used to create it. Additionally, he advised us to make a request for verification of the technological development model, establishing that it is a closed production process.
Representative of Galiltec S.A. Ecuador, an agro-biotech company specialized in the production of micropropagation plants, providing elite banana and plantain material in Ecuador.
The project had already migrated its focus to the dsRNA-based biopesticide with less relevance to the diagnostic approach. As a result of the consultancy, we found that productivity is the main factor that clients consult when purchasing banana plant material, even above tolerance and/or resistance to pests or diseases. Celso also noted that biosecurity regulations to prevent the entry of Foc TR4 at border crossings and at the entrance to banana farms have been significantly relaxed, so our product may become more interesting in the market in the future.
Jhonny is an Industrial Systems Engineer with certification in Project Management at the Polytechnic University of Madrid. Entrepreneur with more than seven years of experience in AgroTech, CEO of AgroScan a Startup dedicated to research, development, and innovation in the agricultural sector.
The team proposed the development of a detection device for FOC TR4, using the LAMP molecular technique. The result would be issued by a color change, based on the prediction of a neural network model, which would be reported in real-time through a mobile application. As a result, the time required to diagnose a banana plantation was questioned, considering that they measure up to more than 100 hectares. And to use neural networks, a considerable volume of plants with the disease is needed. When we saw the difficulties we would have to use neural networks since the fungus is not currently available in the country, therefore we quitted with this idea.
Eduardo is a small banana grower, involved in the process of harvesting plants and the challenges that crops face when a disease attacks them.
Eduardo mentioned to us that the biggest problem for growers is the lack of knowledge of pesticides. For him, it is better to burn affected plants and to use natural fertilizers rather than applying chemical pesticides due to the damage they can produce to the soil and health.
As part of the discussion, we also wanted to know his opinion about GMOs. He told us that he is against these types of products because he thinks that they can attack other plants and this industry is a monopoly that does not really benefit small growers as he.
With this dialogue, we realized the lack of knowledge that some growers have about pesticides and GMOs. Therefore, we encourage future iGEM teams to communicate the importance of synthetic biology and the benefits it could bring to the agricultural sector for pest control.
Davi Schmidt has a master's degree in Agroecology and Rural Development and works in the area of agriculture in partnership with EMBRAPA.
It was mentioned that in the presence of Fusarium in crops, it is difficult to establish a perimeter of crops at risk and it is preferred to quarantine the entire crop. In Brazil, there is a high prevalence of Fusarium, so it is necessary to work on projects for the development of diagnostic systems. He mentioned that the preventive approach could be important for banana industries, but its applicability could be limited.
Methodologies for the application of the product should be established and thought should be given to how its application could be carried out in large extensions of land.
We contacted scientific experts in different fields such as agriculture, plant pathology, synthetic biology, molecular biology, and biotechnology. All of them helped us to understand the behaviour of the fungus we chose to work with (FOC TR4). We learnt about the harmful effects it can produce in banana crops, how microorganisms can be used to obtain biopesticides, the mechanism of interference RNA, how to develop gene synthetic circuits from parts assemblies, etc.
We contacted banana growers to know about all the issues that can affect their crops. How do they fight against pests? The growers told us about the need for effective biopesticides that attack pathogens without harming the crop or the health of the consumers. They also fear for the problems produced by chemical pesticides. Interestingly, they gave us a new perspective from the legal point of view, encouraging us to contact governmental entities that enact environmental regulatory laws to control plagues.
To verify that the different approaches that our project was adopting were in line with the legal framework, we established contact with representatives of the Ministry of the Environment, Water and Ecological Transition . This advice allowed us to modify our initial approach to our idea by taking into account the limitations of our country's regulations, such as the ban on genetically modified organisms (GMOs). In the end, the Ecuadorian authorities were open to integrating synthetic biology as an alternative for the development of the bioeconomy in Ecuador.
Since our project involves the production of a biopesticide, we talked with agrochemical’s suppliers, certified plant material providers, and other products that contribute to banana producers. As a result, our project was modified according to the commercial perspectives of them, and their recommendations for scaling up the project at a commercial level.
We approached scientists in charge of research on pathogens in musaceae. This advice allowed us to orient technical aspects of our project, such as how to administer the dsRNA to the plant, what alternatives are being considered to counteract Foc TR4, among others.
Due to the banana’s commercialization to other countries being one of the principal incomes for Ecuador, we talked with banana exporters to find out how much they know about Fusarium and its harmful effects. We discuss the importance of early low-cost diagnostic systems, preventive measurements against Fusarium, and the impact on their economic activities if the fungus infests their crops.
Integrated Human Practices
Communication
Education & Public Engagement
Inclusivity
Sustainable Development Impact
Entrepreneurship
Human Practices
Our project involves 3 main fields Banana producers, Ecuadorian regulatory entities of Agriculture & Environment, and Academia. We wanted to learn about their realities and provide a solution that meets their demands. This way, our iGEM project raises as an integral and innovative solution for Ecuador.
Overview
We have designed Agrobactory 593 against Fusarium wilt, which is a lethal disease that mainly affects bananas. In Ecuador, banana production is the cornerstone for the country’s economy because it generates thousands of jobs. Ecuador is one of the world's main banana exporters, therefore, the consequences of the pathogen’s arrival into our territory would be devastating for the socio-economic sector. Human Practices and these 3 areas included: analysis of the banana-value chain (production, processing, and marketing), as well as feedback from the legal, environmental, and academic actors.
We held meetings with key players in the agricultural, legal and academic sectors of our country to improve our understanding of the realities faced by banana companies in terms of lack of information and advice on pest control. hese meetings helped us to a better understanding of the necessities of large and small banana producers. In the end, all these experiences encouraged us to create Agrobactory 593 and a product that could be applied to the soil as a preventive measure, but always considering GMOs regulatory restrictions in our country and worldwide.
We also were involved in the development of new legal bases that modify biosafety considerations regarding GMOs in Ecuador. On this page, we'll share what we've learned through working with farmers, agricultural leaders, and academic experts. We also want to show how these exchanges have influenced the development of our project to ensure a solution suitable for Ecuador's reality.
Our Stakeholders
(click on the circle).
Scientific Experts
Banana Producers
Government Entities
Agricultural Inputs
Agricultural Experts
Exporters
Scientific experts
On this part, we will report the insights that interactions with players from the scientific and agricultural world, the academic and the business world have provided us during the entirety of our project . We have compiled here how our initial project idea was shaped by the experience and knowledge from the stakeholders. Click on the circle> .
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Linda Guamán & Carlos Barba.
Synthetic biology and CRISPR-Cas9 experts
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Ediner Fuentes
Responsible for the Virology and Biotechnology Department in the Institute for Health Studies
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Otto Heringer
Entrepreneur of biotechnology projects
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Salome Suarez
PhD student in Plant Pathology at Oklahoma State University.
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Martha Carolina Elizondo
After iGEM Ambassador Coordinator, Master student at Heidelberg University
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Antonio Bustamante
Responsible for the Musaceae Program of the Pichilingue Tropical Station of INIAP.
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Freddy Magdama & Efrén Santos
Experts in Plant Pathology and teachers at the Escuela Superior Politécnica del Litoral (ESPOL)- Ecuador
Banana value chain groups
Our human practices also focused on the banana value chain, which is the set of all the activities necessary to obtain a product (in this case bananas), those that provide added value and also benefit consumers, such as: primary activities, secondary activities, suppliers and exporters.
In this section we will show you the advice we received from each of the experts that make up the banana value chain (click on the circle).
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Jorge Patricio Sánchez Gutiérrez
Group: Midsize producers
Entity: Bananera Pacidel S.A / Association of small banana producers "El Guabo".
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Alexis Vela
Group: Government Entities
Entity: Ministry of Environment, Water and Ecological Transition
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Celso Carranza
Group: Inputs (Certified plant material)
Entity: Galiltec S. A., Ecuador
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Jhonny Villacís Sánchez
Group: Technical Experts
Entity: AgroScan
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Eduardo Clodoveo Moncayo
Group: Small producers
Entity: Rancho San Eduardo
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Davi Schmidt
Group: Large producers
Entity: Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA)
Global Policy Hackathon
During september 24th, 25th and 26th , several experts in local and global policies for importing/exporting materials used in Synthetic biology in the Global Policy Hackathon. Here, we were able to analyze and contrast the current Ecuador’s legislation related to this matter.
The iGEM Ecuador team analyzed “Procedures for importing microorganisms, DNA and RNA”. We found ambiguity in the definitions of "Genetic Material", "Diagnostic Reagents" and "Biological Products", as well as the non-existence of specific laws for the importation of microorganisms, DNA, and RNA, causing the procedure to take a long time which could result in the material being impossible to import or arriving in poor condition.
Our findings led us to propose the use of internationally standardized definitions such as those described by the Food and Drug Administration (FDA) to import procedures from other regulatory bodies and to extrapolate regulations for the importation of genetic material used in synthetic biology as long as the material to be imported was not genetically modified, does not represent a pathological risk and comes from countries belonging to the Andean Community of Nations.
As a result of our participation in the event, we have generated a Overview of the Ecuador's import policies for biological parts, DNA/RNA and microbes.
At the end of the event, each team presented their work during the hackathon and the iGEM Ecuador team received the award for "Best Presentation" and "Policy Overview Runners Up".
References
[1] M. B. García, F. Juca y O. M. Juca, «STUDY OF LINKS OF THE VALUE CHAIN OF BANANAS IN THE PROVINCE OF EL ORO,» Universidad y Sociedad , vol. 8, nº 3, p. 8, 2016.
[2] V. Gurdaswani, S. B. Ghag, y T. R. Ganapathi, “FocSge1 in Fusarium oxysporum f. sp. cubense race 1 is essential for full virulence”, BMC Microbiology, vol. 20, núm. 1, p. 255, ago. 2020, doi: 10.1186/s12866-020-01936-y
