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 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 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 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.

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. 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 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.

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 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.

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 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. 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!

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.

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.

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".

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.

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.

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.

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 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.