UNDERSTANDING THE PROBLEM
Stakeholder Interviews
Before investing our energies in doing wet lab and dry lab work, we wanted to verify that vitamin A deficiency is still a problem. We talked to people who work directly with vitamin A deficiency: nutritionists, public health specialists, anthropologists, and doctors. We created conversation about the problems with past attempts in vitamin A supplementation programs, what the supplementation process is like, the current status of vitamin A internationally and countries they are in, and the bottlenecks in the supplementation process.
Lourlin Ugdiman
Government Public Health & Nutrition Worker
Lourlin is a Public Health & Nutrition Worker working for the government in Arayat, Philippines. It was crucial for us to understand the status of vitamin A deficiency in the Philippines, which was once a hotspot for VAD-caused child blindness. Lourlin also provided us insights on how supplements from the government reach the community.
Vitamin A is supplied every 6 months in the community every 6 months. The government provides the supplements while healthcare and nutrition workers like Lourlin provide them in schools, municipal centers, clinics, and individual houses that have difficulty going to these centers. The supplements they provide are sourced externally, such as India and China.
In our interview, Lourlin says “We supply vitamin A every 6 months at April and October. That (picture) is given by the Rural Health Unit in the Department of Health. The 200,000 IU we give to children 12-59 months, while the 100,000 IU which is blue (bottle) we give to kids 9 to 11 months. It’s not (VAD cases) like before because now the government provides the supplements. But it's still not sufficient because of inadequate sustenance in their own families. ” -((translated from Kapampangan)
Dr. Charles Mather
Medical Anthropologist
Dr. Charles Mather is a professor of social and cultural anthropology, sub-Saharan anthropology, and medical anthropology at the University of Calgary. As an individual with considerable experience in sub-Saharan cultures and medical systems, we contacted Dr. Mather for insight into how sub-Saharan regions of Africa are affected by vitamin A deficiency, and how they may go about implementing our solution.
Dr. Mather also provided some insight into the cultural associations surrounding blindness, stating that spiritual healing medicines and shrines play a big role in Ghanaian culture. However, he cited the Ghanaian people as highly pluralistic and open-minded, stating that they are willing to adopt new beneficial technologies and medicines as they become available. In order to implement a solution in a long-term and sustainable manner, he recommends beginning at the village level. He said there are also a variety of health conditions and dietary problems asides from vitamin A deficiency such as rickets and anemia that the people there are facing since many of those he has worked with were from low-income families.
Dr. Sanou Dia
Public Health Nutritionist
Dr. Sanou is a public health nutritionist who has worked with UNICEF, National Public Health Laboratory, and National Nutrition Center in Burkina Faso. He received a Ph.D. in Nutrition from Laval University and completed his postdoctoral training in aboriginal health at the University of Alberta. A graduate from the University of Ouagadougou (B.Sc. and M.Sc.), Professor Sanou also holds a Certificate in information technology and nutrition from the Swedish University of Agricultural Sciences.
The goal of our meeting to understand what bigger organizations such as UNICEF do to fight VAD. We also wanted to talk to him more specifically about the steps we would need to take from creating a product in the lab to its deployment in communities. He gave us insights about the vitamin A fiasco and the reasons why projects such as golden rice and other biofortified crops don’t work. This includes farmers preferring earning money for planting exportable crops over biofortified ones. Additionally, carotenoid to retinol bioconversion in the human body is minimal since the crops do not have a steady supply and take long to grow. With biofortification still being a new technology and thus making a very small part of the diet, Dr. Sanou says that having a regular supply of vitamin A rather than twice a year would be “much better”.
He works with communities to have “micronutrient week” or “vitamin A week” where healthcare workers mass supply vitamin A and educate them about its importance in diet. His concern though, is that even with more supplements sufficient for more than 100% of the country, there will still be individuals who will never access them due to issues such as distance and lack of proper roads and infrastructures.
We learned that universal supplementation of vitamin A for children under 2 years of age is coupled with deworming. This is because intestinal parasites perforate the intestines and prevent the maximal absorption of the vitamin.
Dr. Sanou explained that moving to a larger scale for supplementation, we would need a technologically feasible and impactful project. After obtaining replicates of our results, he advised us to reach out to funding institutions Canadian Institutes of Health Research and produce scientifically sound data. Then, we would work on the implementation and scaling up of the project before reaching out to humanitarian organizations and larger international funding institutes for distribution of our product.
Dr. Warren Wilson
Biological Anthropologist
Dr Warren Wilson is biological anthropologist from U of C who tries to understand what influences health and nutrition in developing countries. His research has brought him to Colombia, Guyana, Tanzania, Nicaragua, and right here in Canada, where he has studied health, nutrition, and the social variables that predict them, particularly in children. By living on-site and immersing himself in the societies he studies, he and other biological anthropologists develop deep understandings of the human factors that affect a population’s health, including VAD.
From the start, Dr. Wilson urged us to consider very deeply the convoluted human realities and causes of the problems we sought to solve. His experiences with humanitarian projects had taught him that such operations, if done thoughtlessly, could cause far more harm than good. As one example, he told us of a humanitarian project which built a fishing pond in a village in the Amazon Rainforest for the benefit of the villagers. However, due to lack of understanding, they failed to consider the fact that the village already had ample access to fish from the local river, so their efforts were useless. Even worse, the standing water allowed mosquitoes to reproduce and spread malaria in their village, which had previously been malaria-free.
He taught us that the way to avoid this pitfall was essentially extremely robust human practices. We needed to consult many people who had personal experience and stakes in the area. Only those people could tell us whether our idea was needed, whether it would be accepted, and whether it would work. He stated that with many kinds of aid intervention, including vitamin A supplementation, one of the greatest flaws is their lack of ability to continue once the administration is gone. If an external force provides all of the resources, as soon as they cease to provide that support, the problems simply return to their original state. Therefore these programs only build reliance on external help.
Finally, he also brought to light the severe lack of up-to-date VAD population data. This is due to the necessity of drawing blood and physically shipping it often hundreds of kilometres or more to be analysed in a lab, placing serious constraints on practicality.
Dharamwati is an Accredited Social Health Activist (ASHA) worker in the Health Department of Delhi serving farming communities near Delhi. As an ASHA worker, she visits houses and conducts surveys about the health and economic status of the household. The ASHA workers in coordination with Aanganwadi workers, enroll the children and keep regular tab on their weight, physical and mental growth by providing them with nutritious food and involving them in various constructive and educative activities.
We learned that they perform diagnostic tests of the children done, in order to identify and monitor vitamin, calcium and iron deficiencies. They also help enroll expecting women in Aanganwadis, so that proper food and economic help (Government Sponsored) can be provided to them.
Dharamwati said that 20-25% of children in her area suffer from vitamin A deficiency. Together with ASHA and the government, they provide vitamin A drops every six months to children from 9 months to 5 years of age. They also counsel mothers to incorporate vitamin A rich foods in their diet.
She brought up her concern of the non-availability of the vitamin A syrup sometimes, and community members resisting administration of the vitamin to their children. This inspired us to develop a sustainable synthetic biology solution to vitamin A deficiency by using plentiful resources such as agricultural waste, that would normally be discarded.
What did we discover?
Current State of Vitamin A Deficiency
Our HP contacts who work in the Philippines, Ghana, India, and Burkina Faso told us that VAD still affects a significant percentage of the children in their countries. The government plays a big role in promotion of growing beta-carotene biofortified crops and supplying vitamin A (retinol) supplement capsules. However, these efforts are not enough.
Lack of Sustainable Solutions
Many institutions from western companies have provided VAD-vulnerable countries with resources to combat the problem, but we learned that they don’t work well because these institutions do not leave infrastructure for the people to work with. As a result, their efforts go in vain as soon as they leave the country and stop providing resources. Many of these efforts also do not consider the culture and available resources in the country they are helping. Lastly, the money has to come from someone’s pocket. Even with cheap vitamin A capsule prices, transportation and distribution costs can still be economically straining. Most importantly, the people we are working with do not want external aid enforced on them. They want something sustainable-- something THEY can have ownership and accountability of.
Minimal Community-based Implementation
Past approaches such as the Golden Rice project and biofortification of other staple crops through genetic engineering did not work well because plants need months to grow and farmers would suffer economic losses because the biofortified crops can contaminate their non-fortified crops and prevent its export. Additionally, vitamin A supplementation through gel capsules is only done biannually. Health and nutritional organizations allot for more than 100% coverage of supplements, but social workers and mobile nurses are not always able to reach rural areas.
Inadequate Access to Healthcare
Vitamin A supplementation is not always effective because children's intestines are often perforated by worms and parasites due to lack of proper footwear and access to clean water in these areas. As a result, deworming using Albendazole is coupled with the vitamin’s supplementation. Asides from these initiatives, we were surprised to hear that diagnostic methods for VAD are based on height, sight, and academic performance tests rather than laboratory testing. This explains the sparse data on Vitamin A deficiency by the WHO, which has not been updated internationally since 2005. From Dr. Sanou’s Public Health perspective, these tests are inaccurate and are not a good representative of the actual numbers on vitamin A deficiency.
IDEATE
Propose Solutions
Having identified key problems in the vitamin A supplementation distribution, our team started brainstorming ideas. Upon careful consideration of our stakeholders’ needs, we decided on using a chassis producing β-carotene targeted for consumption, like nutritional yeast. Upon conducting literature review, we decided on working with Rhodotorula toruloides, a red oleaginous yeast which naturally produces β-carotene. Then, we would genetically modify it with a series of cellulases to use agricultural waste as a feedstock. In our first plan, for added nutritional benefits, we would engineer two additional genes to our chassis for expression of EPA, an omega-3 fatty acid known to have cognitive benefits. A light-inducible kill switch would also be added as a biocontainment measure. For added safety features and optimized growth conditions, our yeast would be grown in a laboratory bioreactor. With these attributes, the yeast could be grown in a community center, clinics, or dispensaries-- improving vitamin coverage and delivery in a sustainable manner.
To learn more about these solutions, please click the buttons below.
Verifying Need
With a proposal on hand, we talked to our end users to verify its need and discuss its implementation in their workplace and communities. We talked to healthcare workers and nutritionists in rural areas in South Asia, Sub-Saharan Africa, and Southeast Asia which are among the most VAD-vulnerable areas.
Lourlin Ugdiman
Public Health & Nutrition
In an interview, Lourlin told us that despite their supplementation efforts towards children from 9 months to 59 months of age, it is often not enough due to insufficient food and nutrition in their own families. When we asked her about the possibility of setting up a field-adapted bioreactor (FAB) in her village community, she said "Kung may budget, gamit, at may personnel, para sa pag-gawa ng vitamin A, bakit hindi? Willing makipagtulungan ang gobyerno para sa ikauunlad at kapakanan ng kalusugan ng mga tao sa kumunidad.” which translates to "If there are finances, resources, and personnel, why not? The government is willing to work together as long as it’s for the development and welfare of the people and community’s health."
Tyler Warnock
Medical School Student
Tyler Warnock is a medical school student at the University of Calgary. He has worked with Healthy Child Uganda and Mama na Mtoto in Tanzania in developing their community health worker model. Part of his duties were seeing if training programs were having an effect and studying the narratives of people involved. Some of the main issues their team faced were the lack of training and equipment in rural clinics and dispensaries and transportation due to lack of infrastructure. Because of this they had a hard time transporting children and pregnant mothers to clinics and administering vaccines, causing preventable illness and infections.
Tyler also emphasized the importance of ethical practices in research. He informed us about the “unethical practice in global health” where North American researchers would come in to collect data and leave the community without any form of infrastructure, causing a division of community and researchers due to distrust. This, in combination with feedback from Dr. Sanou and Dr. Wilson that current testing was impractical and inaccessible, encouraged us to work on a portable diagnostic device for vitamin A levels in the blood (the Randle’s Cell
). This would enable us and the community to determine whether our efforts were making a difference, including them in the process. Ultimately, Tyler said that building trust and logistics on how to import the bioreactor and how the communities can implement it in the long term should be a big focus of our project.
Tyler also gave us insights on the East African Diet, which comprises mainly starches such as bananas and millet flour. This inspired us to work on more recipes for our implementation cookbook.
Dr. Dia Sanou
Public Health Nutritionist
Upon pitching our proposal to Dr. Sanou, he thought it was a very innovative technology and appreciated the idea. He warned us that the challenge was not having the yeast produce vitamin A, but “how do you take the yeast to consumers, to the target group?”. Yeast is most often used in products like yogurts and breads. He suggested following the yogurt-making model in Women’s centers where women are trained to make yogurt which is distributed to schools, giving rise to employment while empowering women. Dr. Sanou emphasized the importance of incorporating it into food rather than consuming the product by itself because of its flavour and nature among many factors. He thinks that the best way for its incorporation would be through yogurt, porridge, traditional cakes, and cereals.
Dr. Sanou considers omega-3 production an excellent idea, but intestinal parasites might altogether impede our efforts in vitamin A and omega-3 supplementation.
Dr. Samir Gupta
Respirologist & Co-founder, GPS Health Program
Dr. Gupta is a Respirologist in the University of Toronto and is one of the co-founders of the Health Program at the Global Pathway School in India, which was developed by Canadians to help Indian students living below the poverty-line. His background as a medical doctor and a leader in developing the health program in the school was important for us learning about implementation and working in international communities.
We learned about their application of a 15-point plan which includes providing resources from workshops on life skills, immunization, to counseling parents and children. We were most interested in their feeding the students meals in school, and supplying vitamin A and Albendazole for deworming as it relates directly to our project.
Dr. Gupta expressed interest in our project, especially since deworming and vitamin A supplementation were components of the Global Pathway School’s Health Program. However, as a doctor, he wanted more emphasis on its safety and technical aspects. He told us to look more into the pharmaceutical properties of thymol as an antiparasitic, especially its efficacy compared to current drugs like Albendazole which are highly effective. With regards to our field-adapted bioreactor, he said that if “there’s an actual way to do it, that would be more preferable to our kids too.”.
WNYEC Uganda
Nonprofit Organization
The West Nile Youth Empowerment Centre is a youth-led nonprofit organization empowering youth voices to improve services, livelihood, programs, and policy. It increases opportunities for people’s choices of leading a long, healthy and creative life with a decent standard of living, and the freedom and dignity to make decisions for their well-being.
Their efforts are mainly targeted towards combating poverty as it is multidimensional, involving ill-health, illiteracy, lack of access to basic social services, and little opportunity to participate in the processes that influence and improve people’s lives.
We reached out to the nonprofit because we wanted to build a partnership where we can test the Oviita system, while incorporating feedback from our target communities in its design. We talked to Nadia Izzi- the programs officer of WNYEC- who liked the idea because it would offer employment opportunities to the community while addressing a healthcare concern. With this partnership, we would be trained with the different cultures of the populations they are dealing with, while we train their staff how to operate the FAB bioreactor. After their staff is trained, they would embrace leadership roles and promote microenterprises for products such as yogurt fortified with Oviita.
To learn more about West Nile Youth Empowerment Centre please visit their Facebook page.
Dharamwati thought that our proposal was an innovative solution, as it would ensure less dependency on supplements and Government Supplies while providing regular and constant supply of Vitamin A for the whole family. Since people would be preparing the yeast themselves they would be more confident about its efficacy. This brings up an interesting point about the thoughts of community members compared to medical doctors who are more concerned with safety around dose regulations.
She thought our solution would be adoptable since our field-adapted bioreactor materials are easily accessible in their community, and allotting an hour per week to prepare the yeast would not pose a problem. This is something their community would be willing to pursue since they are aware of the consequences of malnutrition for their children, and they know that these issues, if tackled early and naturally, would ensure a better future for their family. It is also a great incentive for ladies in the community who would love to earn extra money.
As an ASHA worker, Dharamwati thought it would be best to mix yeast with food wherever it is being prepared so that the kids get Vitamin A rich diet consistently. Dharamwati expressed great interest in training ladies, saying, “We can train and educate ladies in such a way that they not only prepare yeast for their community, but also train women of other communities and thus this can be developed into an environment-friendly and Health-Boosting Small Scale Industry.
The feedback we received from our stakeholders was that a bioreactor capable of producing a steady supply of vitamin-A producing yeast would be much better than waiting for biannual distribution of the vitamin. Omega-3 fatty acids are an emerging essential nutrient with numerous health benefits which make its production favourable.
Dr. Sanou mentioned that even with omega-3 supplementation, intestinal parasites will still pose a problem in preventing the absorption of not only vitamin A but the omega-3 fatty acids too. Because of this, our team decided to change the production of omega-3 to that of thymol, an anthelmintic compound found in thyme leaves. The compound is shown to critically damage Caenorhabditis elegans and its eggs, which is the gold standard in anthelmintic drug testing. While this is not a cure to intestinal parasites, it can mitigate intestinal damage and improve general micronutrient absorption until drugs such as Albendazole are distributed, even without access to clean water or proper footwear.
In places like the Philippines, India, Burkina Faso, and Uganda, people would have events like “nutrition week” or “micronutrient week” dedicated to diagnosing VAD, deworming using Albendazole, supplying vitamin A, and also informing parents and children about the importance of incorporating vitamin A in the diet. Dharamwati thinks that having a communal FAB bioreactor would be better than individual ones for homes as it would be built with shared investment, and encourage community participation especially in events such as nutrition week.
We talked to Tyler Warnock, a medical school student who has worked with Healthy Child Uganda and Mama na Mtoto in Tanzania about his experience with global health work in Uganda, and how we could apply them towards our bioreactor and project implementation. He cautioned that making a difference is all about relationship building, and its absence would make our project implementation very difficult and “ unethical”. Many western people come in to collect data and leave without infrastructure, causing a divide between them and the local people.
With unethical implementation and inaccurate testing in mind, our team worked on the Randle Cell Testing Device which detects vitamin A levels in the blood. This is important because it helps us build a more accurate picture of VAD in our target areas, and informs us and our end-users whether our solution is making a difference.
To learn more about the Randle Cell Testing Device and Thymol Production, please click the buttons below.
We also talked to Dr. Samir Gupta, one of the co-founders of the Health Program at the Global Pathway School in India, which was developed by Canadians to help Indians students living below the poverty-line. We learned about their application of a 15-point plan which includes feeding the students meals in school, and supplying vitamin A and Albendazole for deworming. We asked him how he and his team successfully built this community without being culturally insensitive. He said that it is important to employ local people in leadership roles and in positions which enable community members to connect with them around economic and social problems. For the implementation of our yeast, he suggested considering schools for distribution since kids would be in one location being fed.
Our HP contacts agree that having microenterprises linked to Oviita would make our project more self-sustaining as it would train the local people while providing empowerment and employment, especially to women. Taking Dr. Gupta’s feedback in consideration, we partnered with the West Nile Youth Empowerment Centre (WNYEC) in Uganda which empowers youth through cultural and entrepreneurial training across eight different regions in Uganda. After explaining our project and its microenterprise opportunities, Nadia Iddi, the programs officer of WNYEC, said that this would be a great idea as it would create employment opportunities in their area. As long as our team provided them the initial training, they would also train their community members in how to operate our bioreactor, creating products like yoghurt fortified with Oviita, and educate people with the importance of vitamin A in the diet. Once the proposed training model is set in place, our partnership would expand to nearby health clinics around the area. Nadia informed us that the general perception for genetically modified organisms in Uganda is negative, but that should not be a threat with its monetary and health benefits. Over the next year, we will be working closely with them to see how we could best integrate the Oviita system tailored to their resources and culture.
The graphic above represents the Oviita system we've build hand-in-hand (virtually) with our end-users after various iterations. Now that we had a stakeholder-approved proposal, it was time to figure out how to make the science work.
DESIGN SOLUTIONS
Expert Consultations
CELLULASE ENGINEERING
Vitamin A supplements are typically provided or sold by foreign bodies. If the supply-chain are disrupted, VAD-struck countries would not receive the vitamin. Unfortunately, this is the case with many humanitarian organizations such as Save The Children International, which halted supplement and material donations to vulnerable countries due to COVID-19 (Personal Communications, Save the Children US). Thus, we needed to come up with a self-sustaining solution that the community could maintain. Many of these communities grow crops for a living, so we thought to engineer cellulases into our yeast. Since our team has no experience working with Yarrowia lipolytica, we needed to know what considerations were required for our system.
Dr. Raymond J. Turner
Biochemistry Professor
Dr. Raymond Turner is a professor and researcher specializing in microbial physiology and biochemistry. He provided extensive feedback at the Faculty Talk, answering many questions about how best to engineer our gene cassettes. He agreed with Trevor Randall that we should use Gibson assembly and combine multiple cassettes in E. coli into one single cassette for use in Y. lipolytica
Dr. Charles Mather
Medical Anthropologist
Dr. Mather- a medical and sub-Saharan African anthropologist- agreed that an edible yeast could be an excellent way to to mitigate VAD. However, he informed us that in West Africa where he has lived and worked, the people at risk from VAD do not have a lot of excess spending money to support yeast, and that ongoing sugar purchases to fuel them would be too expensive to be viable. However, he stated that people repurpose agricultural waste in many ways, and already have extensive knowledge in how best to process and prepare plant biomass for microbial digestion in household beer-brewing. Therefore, he believed that basing the yeast on an agricultural waste food source would allow the technology to be accepted and embraced.
Dr. Rodrigo Ledesma-Amaro
Bioengineering
Dr. Rodrigo Ledesma-Amaro is a researcher in synthetic biology and metabolic engineering, with a focus on Y. lipolytica
metabolism and use as a synthetic biology chassis. He provided invaluable advice to us in how best to work with Y. lipolytica, as well as providing protocols for transformation and preparation of chemically competent cells. Vitally, he also provided us with a strain of Y. lipolytica already engineered by his lab to produce beta-carotene.
Trevor Randall
Systems Biology
Trevor Randall is a PhD student with a project in biochemistry and systems biology. He advised the engineering of our cellulase gene constructs. Specifically, he recommended that Gibson Assembly would work best for our purposes, and that we should combine multiple cellulase genes into one cassette for transformation into Y. lipolytica.
THYMOL PRODUCTION
Vitamin A supplementation is coupled with deworming with compounds like Albendazole twice a year. This is because intestinal parasites perforate the intestines and prevent the maximal absorption of vitamin A and other micronutrients. Despite biannual deworming, lack of access to clean water and proper footwear only causes intestinal parasites to return. Additionally, these worms may also develop drug resistance especially since anthelmintic and deworming agents commercially available are limited. To alleviate intestinal damage, we decided to genetically engineer our yeast to produce thymol, a compound found in thyme leaves. Thymol has been shown to have anthelmintic properties against Caenorhabditis elegans and their eggs.
Our team had no clue how anthelmintics worked, or how drugs like Albendazole transition from the laboratory to schools. To find out more, we talked to a molecular and developmental geneticist, a doctor, and a parasitologist.
Paul E. Mains
Professor of Biochemistry & Molecular Biology
Dr. Mains is a biochemistry and molecular biology professor at the University of Calgary. He works with the resistance of worms to the anthelmintic benzimidazole, and developing Caenorhabditis elegans embryos. His background was vital for planning the experimental design of thymol production.
Dr. Mains said there was merit to producing thymol in our yeast because 1-2 billion people are infected by nematodes, mainly school-age children. There are effective treatments, but based upon his experience mainly with sheep parasites, resistance is inevitable so new treatments should be kept on the radar.
Dr. Mains thoughtC. elegans might not be able to consume the yeast because of its thick cell wall, and advised us to perform a simple experiment where we place non-engineered yeast on a petri-dish with worms first and see if they an use the yeast as an energy source. Additionally, he said we shouldn’t place C. elegans on a yeast plate but put yeast in C. elegansEscherichia coli if thymol is excreted, or add thymol to E. coli and feed that to the worms.
We thank Dr. Paul for generously offering to pick worms and perform thymol tests on C. elegans
Dr. Samir Gupta
Respiratologist & GPS Health Program Co-founder
Dr. Gupta raised concerns around the safety and efficacy of thymol compared to existing drugs like Albendazole. While we might think that having thymol would be more beneficial than waiting for biannual deworming, we should still prioritize thinking about its safety.
We asked Dr. Gupta what kinds of regulations we might need to consider if we were to deploy thymol from the laboratory to an edible supplement. We found out that pharmaceutical trials and supplement trials are done differently. Depending on the character of thymol, we might be able to market our yeast as a supplement rather than a pharmaceutical. This would be faster and more cost-effective than pharmaceutical trials which involve larger human trials. He also agreed that probiotics might be a good parallel for regulation of our product, but its genetic modification might change the regulatory system for Oviita.
Dr. John Gilleard
Professor of Parasitology
Dr. John Gilleard is a professor of parasitology in the Comparative Biology and Experimental Medicine department at the University of Calgary. His research interests lie in field of drug resistance in parasites. Dr. Gilleard also has an interest in the development of novel diagnostic approaches for parasitic infections and anti-parasitic drug resistance, particularly for cattle parasites.
Talking to Dr. Gilleard, we learned that there are only two drug classes that have been in use since the 1980s. On the livestock side, it’s used as a preventative measure with monthly treatments in more temperate regions. Because of that, resistances emerge. In most worm cases in sheep such as in Australia, the worms are resistant to almost every other anthelmintic drug. For cattle and horses, drug resistance is starting to be more widespread too.
Dr. Gilleard helped us understand the severity of the problem in sheep models. He said that approximates 10,000 worms in one sheep which produces 4,000 eggs each. In a field, there could be as much as millions of worms depending on the size of the pasture. Since these worms reproduce sexually, resistances such as those caused by single-nucleotide polymorphisms are passed to their offspring. In other words, there could be more genotypes in a pasture than people in the world. As a result, existing drugs only exhibit partial efficacy. It is important that we study sheep and cattle models because the same outcome is expected to arise in humans, as anthelmintics in humans were first tested in livestock models.
Dr. Gilleard said that it is very difficult for companies to invest in drug development for anthelmintics in livestock because of costs associated with research and development. Therefore, developing an anthelmintic compound that can be used in rotation with current anthelmintics can help alleviate the problem.
BIOCONTAINMENT MEASURES
A synthetic biology project without a biocontainment strategy would simply be irresponsible. If we wanted our yeast to be deployed outside a lab setting and grown in community FAB bioreactors, we needed to observe extra precautions, making sure we don't do ecological damage to nature. We initially played around with the idea of having a light-inducible kill-switch system, but we weren't sure if that was sufficient.
Thus, we talked to a series of biochemists and molecular biologists who knew a thing or two about effective biocontainment strategies, before finally deciding on an auxotrophic co-culture system.
Dr. Marija Drikic
Metabolomics Research Assistant
Dr. Marija Drikic is a Metabolomics Research Assistant currently working with the Lewis Research Group.
Dr. Drikic helped us during our journey of developing an effective biocontainment strategy and walked us through the many different iterations of the project we had. To begin, she helped us confirm whether our ideas were worth following up on. For example, one of the initial ideas for a biocontainment strategy involved using a light-induced killswitch system to express the Cas9 enzyme such that it would target essential genes of the yeast, killing it. Dr. Drikic’s concerns with this system ultimately lead us to abandoning it to focus on different methods, such as using nucleases to kill a cell, then using an antitoxin-toxin system, before finally deciding upon an auxotrophic co-culture.
Dr. Drikic was also able to help us refine our experimental design for biocontainment, ensuring that the protocols would isolate the variables we were trying to determine. Her expertise aided us in understanding research concepts we came across and how specific protocols work and are carried out. Dr. Drikic was also able to connect us with other HP contacts more suited to specific parts of the project.
Dr. Gordon Chua
Associate Professor
of Integrative Cell Biology
Dr. Gordon Chua is an Associate Professor in the Biological Sciences department at the University of Calgary.
After we had conceived the auxotrophic co-culture biocontainment system we are currently working on, we contacted Dr. Chua for advice and criticisms of this method. Before discussing with Dr. Chua, our co-culture system was developed with four strains in mind. Dr. Chua’s concerns with the system being too complex led to us redesigning the co-culture with two strains in mind.
He eased some of our concerns regarding the auxotrophic co-culture system, mentioning that the possibility of horizontal gene transfer within the co-culture would be very low, there would be little chance of both strains to escape at the same time, described how we could go about making our own auxotrophic strains, and provided direction for the overproducing and auxotrophic amino acid combinations we could use in the system.
Dr. Chua was also able to connect us with other HP contacts, such as Dr. Peter Facchini.
Dr. Vanina Zaremberg
Associate Professor of Biochemistry
As this year’s project is focused on engineering yeast, we needed to understand experimental design in yeast and the protocols involved. This is where Dr. Zaremberg came in, graciously providing us with her expertise, support, and a collection of yeast protocols that we could follow.
We spoke to Dr. Zaremberg back when we were still undecided on a biocontainment system. At this point, we were looking into using Cas9 as a gene of interest that would be inducibly produced and kill the yeast cell. Her main concerns were about the fact that we would have to find essential yeast-specific genes that this enzyme would target, without alternative pathways allowing for the cell to survive. During this time, we were also looking into yeast toxins other than that may be expressed. Dr. Zaremberg advised that we should try to control both expression of the yeast toxin and the transcription factor that activates it, as this was a system seen in bacteria that mitigates leaky expression. She also mentioned that using yeast toxins may not be the most effective biocontainment system.
After looking into Dr. Zaremberg’s comments, we were having difficulty developing a biocontainment system that hit all the marks. Comments from other contacts, such as during our Faculty Talk, put the nail in the coffin. Back to the drawing board it was!
Dr. Robert Mayall
Co-Founder and CTO
of FREDSense Technologies
Dr. Robert Mayall first spoke to us about our biocontainment plans after we had presented the light-inducible kill switch at our Faculty Talk.
He mentioned that while kill switches can be efficient, they can also be mutated out of an organism after many generations. On the other hand, auxotrophy is a more stable system and there were already auxotrophic strains of Saccharomyces cerevisiae available. He also cautioned us about using environmental cues to induce a kill-switch, as these cues may be more common than we were anticipating, leading to premature cell death. Dr. Mayall’s expertise completely changed the course of our project, pushing us towards using an auxotrophic co-culture system, which became our final biocontainment method!
Dr. Peter Facchini
Biochemistry Professor
and CSO of MagicMed Industries
Dr. Facchini convinced us to start with a proof of concept system in S. cerevisiae before we started engineering Y. lipolytica to develop an auxotrophic co-culture system. S. cerevisiae was a model yeast organism, so many tools for genetically modifying the chassis were already available, such as auxotrophic strains and overproducing plasmids. We would save time and energy if we had determined whether this system would work in an easy-to-use organism first. On the other hand, if we had jumped directly into using Y. lipolytica, we would first have to develop auxotrophic strains and build overproducing constructs. Ultimately, this would have taken time, and our results would be much less conclusive than they would be if we used a proof of concept system.
Dr. Facchini also helped provide direction for co-culturing experiments, stating that we would want to look into the relative abundance of the two different strains when they were grown together. In the end, this is exactly what we did!
BIOREACTOR DESIGN
Growing conditions for our yeast must be regulated so we can maintain sufficient beta-carotene production and control beta-carotene doses for consumption. Proper design is also needed to make sure our yeast is contained in a closed-system, preventing accidental ecological damage.
Dr. Raymond J. Turner
Biochemistry Professor
Dr. Turner has years of experience working with scaling up biotech projects and working with bioreactors. During the initial phase of our bioreactor design, he told us that airlift bioreactors were common. For home reactors, people would traditionally use anaerobic fermenters such as kimchi fermenting pots. Since our yeast was an obligate aerobe however, this would be easily solved with an air pump which can be powered through solar energy where electricity is scarce. He also provided inputs on how a bioreactor might look like for those that involve multiple strains of microbes.
Dr. Charles Mather
Medical Anthropologist
Being an anthropologist who has lived and worked in Ghana, Dr. Mather had a lot of inputs to share about our bioreactor’s integration into households and communities. He thought that Northern Ghana specifically would be a good place to start, as the people there “will take initiative with regards to technologies that will have a positive impact”.
Through him we were informed of Northern Ghana’s active beer-brewing industry which could ease the implementation of our field-adapted bioreactor, by potentially utilizing the terracotta pots, biomass processing, and cultural infrastructure they already use for brewing. People in this area reuse everything, including agricultural waste for green manure or silage. Dr. Mather was concerned that some areas might not have electricity, but suggested getting air pumps with solar panels to run the system. He also told us about the temperatures and conditions in Ghana which might affect our yeast growth and protein activity, which inspired us to engineer our cellulases to be more stable.
Juan Sebastian Alvarez
Chemical Engineer
Mr. Alvarez is currently on an internship with FREDSense Biotech during his weekdays. On the weekends he is Chief Strategy Officer for yOIL Technology, a biotech of his own. This man knows lives and breathes DIY synbio, which made him the perfect contact for designing our various bioreactors.
He was an absolute blessing of a resource throughout our bioreactor design process. With his aid, we were able to gain an understanding of the workflows and considerations required to construct a cell growth model. He gently guided us in procuring literature values for our models, ensuring we were always on the right track. This ultimately helped the design and experimentation of our wetlab in their interactions with the bioreactors. Many words come to mind when describing Sebastian Alvarez. If we were forced to describe him with 7 words, they would have to be that he is "both passionate and a darn good engineer".
Trevor Randall
Systems Biology
Dr. Gijs van Rooijen is currently the Chief Scientific Officer of Genome Alberta. He obtained his MSc in Molecular Sciences from the Agricultural University in Wageningen and a PhD in Plant Molecular Biology from the University of Calgary. Dr. Gijs worked for SemBioSys Genetics and (co)authored and co(invented) 25 issued patents in the US.
In our brief conversation with Dr. Gijs, he thought that the project was of great potential and humanitarian aspects. Being an expert in biotechnological implementations, he said that the best way to implement a future in-home individual FAB bioreactor would be to keep the design as simple as possible so our end-users can integrate them in daily life more easily. Additionally, he connected us to the Global Institute of Food Safety to talk about the future directions and potential partnership with our project.
RANDLE CELL TESTING DEVICE
Available data on VAD is obsolete, dating back to 2005. This data was not obtained through testing of retinol concentrations in the blood, but through a series of diagnostic tests scrutinizing vision, height, and academic performance. For Oviita to really make a difference, we need to know whether our supplementation project is needed by our end-users. This is why we created the Randle's Cell Testing Device, a field-based test which utilizes aptamers to quantify retinol binding protein concentrations in the blood. These concentrations can then be correlated to Vitamin A concentrations, enabling communities to detect VAD effectively.
We reached out to professionals from various engineering principles to help the device come to fruition.
Dr. Christopher Naugler
Pathology Informatics
Chris Naugler, MD, is an expert in pathology informatics, and is a recognized leader nationally having authored five medical textbooks. His knowledge of laboratory diagnostic was invaluable to us, as he explained some common tests for micronutrient deficiencies. Dr. Naugler pointed out some issues that we might face from other substances in the blood. He explained some of the FDA and Health Canada approvals that a project such as this one might need. He pointed out that finding the minimum sensitivity needed would be a good start at selecting a diagnostic test design.
Dr. Colin Dalton
CTO, Neuraura Biotech
A senior member of Institute of Electrical and Electronic Engineers and a professor at the University of Calgary, Colin Dalton is a microfabrication expert. He is the Director of the University of Calgary’s Microsystems Hub and is also a co-founder and CTO of Neuraura Biotech Inc. Dr. Dalton opened our eyes to the complexities of making a biosensor, both in and out of the lab. He helped us understand some of the interactions going on at the electrode surface and suggested electrode designs to use. He also informed us of common impedance measurements.
Sultan Khetani
Biosensor Expert
A PhD student at the University of Calgary, Sultan has a background in biosensors and invitro injury modelling. He has experience in developing hand-held, portable biosensors. Sultan gave us valuable information on how aptamer-based biosensors are typically developed. He put into perspective the workflow involved with developing a sensor. He gave us some practical considerations to keep in mind: temperature and shelf-life of our biosensor. He also highlighted immobilization techniques that are used, and explained some of the difficulties with scaling up electrochemical cell biosensors. We valued his input, and these shaped our future directions.
Dr. Robert Mayall
Fredsense Industries
Robert Mayall, PhD, is the CTO and cofounder of FREDsense. He has a decorated background in biosensors and expertise in synthetic biology, nanoscience, electrochemistry, and biosensor development. Robert provided valuable information on the issue of fouling of the electrodes. He also commented on practical considerations for biosensors, and informed us of methods we could use to take these into account. Robert explained some of the software analysis and equivalent circuits that we could use to analyze our impedance data, which allowed us to make sense of our measurements.
Thomas Ljinse
Biomedical Engineer
Thomas Ljinse is A PhD student in Biomedical engineering at the University of Calgary, focusing on microsystems and embedded electronics. He has a plethora of experience in biomedical engineering and design. Thomas gave us invaluable information on aptamer based systems, and informed us of techniques that we could use to measure impedance. He also indicated some of the challenges that we may face taking this design from out of the lab, and into the consumer market. It was Thomas who highlighted the workflow associated with programming an IC and using it for impedance-based analysis.
Evaluate & Iterate on Design
Further Improving Our Design
FACULTY TALK
Shortly after forming our initial proposal, we assembled stakeholders ranging from nutritional anthropologists to electrochemists to evaluate our project proposal. Overall, the feedback we received was promising. However, many questions were raised towards the effectiveness of our light-inducible kill switch, our choice of chassis (Rhodotorula toruloides) and its safety status for consumption, and how we would assemble our constructs for engineering. We were informed that most kill switch systems are susceptible to mutations as there is little reason for the cell to keep the trait. Our system would also have problems if our yeast reaches open waters or goes deep into the soil where light levels vary from minimal to none. We were advised to explore auxotrophy or quorum sensing as an alternative biocontainment strategy. Many of our guests have not heard of our chassis before, and expressed concerns about our ability to engineer it, let alone consume it. We were advised to consider this thoroughly, and use a food-safe yeast such as baker’s yeast instead. While we did not use baker’s yeast, we made the transition to Yarrowia lipolytica, a Generally Recognized As Safe (GRAS)-designated yeast which is oleaginous. Having an oleaginous yeast would aid in vitamin A delivery as beta carotene and retinol are lipophilic. The strain we graciously received from Dr. Ledesma-Amaro’s laboratory in London has been engineered to produce the carotenoid already. Lastly, our attendees educated us about the wonders of Gibson Assembly and how it would save us a lot of trouble while assembling the constructs we needed.
Julygem
We hosted a two-day online meetup in July, where teams from Canada, USA, Taiwan, and Mexico got the chance to share their projects and receive feedback from experienced judges, as well as hear a number of talks by experts in the field that followed the overall theme of translating synthetic biology into the real world. Teams were asked to prepare a 5-7 minute pitch on their project to be presented in front of judges that were not all experienced biologists. Teams that evaluated our pitch though that we should talk to more stakeholders and integrate their needs and feedback to our presentation. The teams also thought we could do a better job of explaining how our solution provides a competitive edge towards existing solutions in vitamin A supplementation, and how both wet lab and dry lab are working together to address the problem more clearly.
iGEM Concordia's Mini Jamboree
We were fortunate to be invited by iGEM Concordia to participate in the Mini Jamboree where we had a friendly competition with four other Canadian teams. The judges thought we were on the right track on working with the local taste palettes and engineering principles. We were cautioned to check our assumptions about the societies we were working with in terms of building materials. Our audience was a bit lost with the values for vitamin A and EPA and how they are linked together, and thought a model on our co-culture switch system would be worth considering. The other teams also agreed that we needed to provide more reasoning why our project was important.
cGEM
We participated at cGEM, a Canada-wide iGEM competition where we presented a poster and a 20-minute project presentation. The area where our judges and fellow teams thought we were lacking the most was showing the integration of our stakeholders throughout the process in our presentation, and whether we have talked to our end-users in vitamin-A deficient areas. At the time, we did not talk to local communities and public health workers so we made sure to do that and reached out to people in India, Philippines, and Uganda. In particular, they were concerned about having a training program or system set in place to ensure the communities knew how our bioreactor would operate, and how the yeast would be properly incorporated into their diets. We were also advised to address any possible obstacles we might face with our solutions
Future Directions
Improving Oviita Beyond the 2020 Jamboree
WORKING WITH WEST NILE YOUTH EMPOWERMENT CENTER IN UGANDA
The West Nile Youth Empowerment Center Uganda is an NGO which empowers youth through cultural and entrepreneurial training across eight different regions in Uganda. This partnership is crucial because we would like to see Oviita’s implementation come to fruition. Dr. Gupta emphasized the importance of having the local people take over leadership roles for Oviita to be successful and self-sustaining. Oviita’s pilot program with the WNYEC is important as other implementations may arise. We will be working closely with them and providing training in the bioreactor’s operation. In exchange, we will also receive training on working with their community and others they are working with. We will be observing models Dr. Sanou recommended, where people get trained in women’s centers on micro entrepreneurial ventures and determine whether our system is working for this environment.
Working with Western Heads East in Waterloo and Ghana
We recently connected with Western Heads East which is a collaboration between Western University staff, students, faculty and African partners using probiotic foods to contribute to health and sustainable development. We talked to Robert Gough, who was more than willing to partner with us and provide information, contacts and resources. He stressed partnerships are huge. Especially on the ground ones and with local universities. He considers public perception of GMOs to be the most challenging aspect of our project. His advice on implementation is to first reach out to a university in Ghana, start a research program to include things like bioreactor, social enterprises and general implementation. During this time, we would also be working through the local regulations. For ethical reasons, any research we would hope to gain from this should be in tangent with local groups. Next we were advised to contact a local organization or hire a local director of operations and then start a specific flagship pilot program. Afterwhich, we would build one bioreactor and then monitor all the data from it and its impact on the community. For Western Heads East, it took four years before they expanded from one kitchen to multiple areas. After sufficient data is collected, we can then use that to market our idea for expansion. For more details, see Entrepreneurship.
Creating an Industrial-Grade Bioreactor with BIONET
As the wiki freezes, BIONET, A Spanish company that designs and supplies bioreactors, reached out to us to provide our team with their support and technologies. Our team will be working on the development phases and equipment requirements in more detail to get this collaboration started after the end of the 2020 Jamboree.
Overview
