Human Practices Breakdown

Our human practices team came up with and implemented a new approach to optimize our project and gather knowledge as new advances were made. This approach involved four stages: Stage 1 - Inquiry, Stage 2 - Formulation, Stage 3 - Integration and Stage 4 - Confirmation. During the first stage, inquiry, our team consulted with individuals who could provide us with guidance on how to structure our team and project. Additionally, we were offered tips on how to maximize the project’s potential from the beginning. The second stage, formulation, took place after we had come up with a potential solution to the issue our project was targeting. This stage involved speaking with experts to gather their input and opinions on our proposed solution. During this stage significant modifications that led to our final project were made. The integration stage or third stage consisted of interviews with the goal of answering specific questions that arose as our project came together. This stage involved thinking about the future of our project and what questions might arise should we pursue our project further. The final stage, confirmation, involved conducting second interviews with experts we had previously interviewed and interviewing others who may provide new ideas on our finalized project. The aim of this stage was to ensure experts and potential stakeholders were satisfied with the modifications made to our project.

INTERVIEWS

Stage 1. Inquiry

Miranda Stahn, Genome Alberta: Expertise in organizing an iGEM team and creating a project. As a former iGEMer and the Program Coordinator at Genome Alberta, Miranda offered our team advice on formulating our project and of potential funding opportunities. She provided us with guidance on what to keep in mind and how to structure our project. We learned of how to best organize our Human Practices subgroup and of what directions might be helpful to take for our project. For example, she suggested our project use a mechanism similar to that of mRNA vaccines. Furthermore, she emphasized that we should focus on the limitations of current solutions and begin thinking of ways to mitigate the stigma on genetic engineering Alan Shapiro: Director of waterNEXT at Foresight Canada, working as a water and sustainability consultant. At the time of our interview with Mr. Shapiro, we were still in the midst of researching potential biological pathways that could be exploited to degrade microcystins. Discussion revolved around stakeholder and public perceptions. We learned that it is crucial to ensure that our solution does more good than harm, even in the early research stages. From there, we thought critically and defined the risks and benefits of potential ideas and weighed them with respect to current solutions. He mentioned that a major hurdle would be the lack of regulations on solutions similar to ours prior to implementation. This might be something that would deter stakeholders. Alan Shapiro introduced us to Fixed Earth Innovations, a company that develops non-traditional bioremediations methods, to discuss this issue further. Shortly after this interview, we decided to employ a mechanism that used antimicrobial peptides. Additionally, this interview is the first where the idea of eventually testing in mesocosm facilities and experimental lakes was discussed.

Stage 2. Formulation

Dr. Greg Pyle, University of Lethbridge: Research in the effects of environmental contaminants on aquatic health. As our original idea and plan at the time of our first interview with Dr. Pyle had been to deliver HPA3NT3-A2 antimicrobial peptides or cyanophage lysozyme to induce cell death in cyanobacteria. However, Dr. Pyle brought up the importance of minimizing off-target effects. This is something we took strongly into consideration, and eventually it led to a major pivot in our project. In addition, he mentioned that we should consider what timing, seasonally, may be best to deliver our system. Discussion on scaling also took place and this information was relayed to our wet lab and modelling teams. He suggested we use modelling tools to investigate the action and structure relationship between the antimicrobial peptides and their receptors on cyanobacteria.

We also learned more on what testing in a mesocosm or experimental facility would look like. He mentioned that this would be crucial as these facilities are similar to natural lake environments and include variables such as the weather. He recommended that we arrange a meeting with experts from a mesocosm facility in Vegreville, Alberta.

Additionally, he brought our attention to the fact that cyanobacteria are primary producers and that not all of them produce toxins. Thus, removal of all cyanobacteria species could result in a bottom-up ecological cascade effect, harming the organisms that feed off of them.

Dr. Brent Selinger, University of Lethbridge: Expertise in Molecular Microbiology.

Similarly to Dr. Pyle, Dr. Selinger was highly concerned about the possibility of non-target effects. He expressed concerns regarding how protein nano compartments would enter cyanobacterial cells and deliver the system. He proposed that we study cyanophage docking and delivery mechanisms. This is something that our modelling team researched and we determined that cyanophages infect a wide range of cyanobacteria. Therefore, they would infect both toxic and non-toxic strains. Thus following this interview, we took a step back to reflect on our project and find a way to make it more specific. Our team eventually settled on the idea of implementing a CRISPR-Cas13a system to ensure specificity. We discussed using an MS2 phage-like particle or cell penetrating peptides as a delivery system. These peptides would be incorporated on our encapsulating protein nanocompartments that would bind to and enter the cyanobacteria.

Stage 3. Integration

Dr. Brian Eaton: Manager of the Environmental Impacts team at InnoTech Alberta. Eager to learn more about mesocosms, we contacted Dr. Eaton, an ecologist with experience researching in this type of experimental system. He explained the operation of mesocosm facilities at Vegreville Alberta and their uses in experimental studies. Some of the challenges that we may encounter since our system cannot by itself, were brought up. For example, we may not obtain enough material for the system to be testable. Thus, prior to scaling our system up we may need to perform toxicity tests to determine the potency of our treatment. Theoretically, each cyanobacteria cell only needs to take up one crRNA to cause cell death. Therefore, we could determine the amount of Cas13a and MlrA enzymes needed to eliminate a given number of cyanobacteria.

Dr. Scott Higgins: Researcher at International Institute for Sustainable Development Experimental Lakes Area (IISD-ELA). We were interested in learning more about scaling up our experiments, thus we contacted Dr. Scott Higgins for the International Institute for Sustainable Development Experimental Lakes Area. Dr. Higgins familiarized us with some of the steps and regulations required for research in an experimental lakes facility. He clarified the timeline for researching in such facilities and informed us of the challenges that might be encountered when creating government regulations for new technologies. We learned that we would need a solid proof of concept in a conventional laboratory that demonstrates a lack of non-target effects, prior to application in an experimental lake. Studies in experimental lakes typically take many years, and the scope of implementation of our proposed treatment would take years to complete. He mentioned that our system would likely need to be applied seasonally, as our solution takes a downstream approach. It was suggested that we couple our system with nutrient reducing strategies to speed up ecosystem recovery. Dr. Higgins emphasized starting with a more simple approach and then gradually expanding. This inspired us to start with targeting the most common strain of cyanobacteria, Microcystis aeruginosa. Once we obtained the desired results, we could eventually look into targeting other strains by including their mRNA sequence as a target in our system.

Dr. Ron Zurawell: Aquatic Scientist at the Resource and Stewardship Division of Alberta Environment and Parks. From speaking with Dr. Zurawell we learned of the governmental regulations that would be imposed on our system should we meet all the testing requirements for it to be used commercially. The policies are quite stringent, thus we decided that we may have to remove water and apply our system in a water treatment facility. While current chemical treatments are effective in eliminating cyanobacteria and the microcystins that some species produce, their use is prohibited in natural bodies of water in Alberta. Although there are fewer regulations for the treatment of man-made bodies of water, such as farm irrigation dugouts, there are still limitations. He also brought awareness to the fact that eliminating cyanobacteria may lead to increased growth of organisms that benefit from a decreased nitrogen to phosphorus ratio. For example, eliminating cyanobacteria from farm water irrigation sources could result in the overgrowth of other harmful algal species, such as Euglena. Which produces toxins that are deleterious to fish and mammals. This would be something that necessitates extensive investigation in the lab. Dr. Charles Holmes, University of Alberta: Research cyanobacteria and microcystins in aquatic ecosystems. Dr. Holmes emphasized the significance of the issue we are attempting to tackle. Not only do Canada and Alberta have a long history of cyanobacteria, toxic microcystins are an issue in the Murray River of Australia and in the Chao Lake of China. These are just a few of the many countries that have problems caused by cyanobacteria. Additionally, we were provided with knowledge on using microcystins in the lab. When we eventually get to the stage of testing microcystins in the wet lab, we will need to keep in mind that small amounts of microcystins can be used as stimulants. Furthermore, microcystins are difficult to eradicate due to their structure. However, our solution takes an approach at the mRNA level, thus degradation is possible. Once we have a workable solution, we were informed that our next steps would be to seek approval from Health Canada. If this is something that we received, potential next steps would be to seek international approval.

Leighton Kolk: CEO of Kolk Farms LTD, and member of the Alberta Cattle Feeders’ Association (ACFA) Mr. Leighton Kolk was more than willing to discuss the impacts of our project on an agricultural and farming level. Mr. Kolk educated us on issues such as fungus overproduction on irrigation/dry land crops within the serial seed retail business, including ways of sustainability to combat these issues. Quite aware of the problem of blue-green algae, Mr. Kolk allowed us to consider any potential issues arising regarding public perception to implement our project; while he stated the importance of taking our time before its usage, he also stressed not taking too long so as not to lose momentum on our objective. As a farmer, we wanted to know how to mitigate the stigmatization around genetic engineering technologies to those unfamiliar on the topic. He stated that though farmers are not overly fond of experimental technologies, it was a necessity in producing high quality food and in the way there are a lot of misconceptions in terms of what farmers do, trusting science and the safety methods and preliminary tests and lab work goes a long way to success.

Stage 4. Confirmation

Timothy Repas: President of Fixed Earth Innovations, Develops Bioremediation treatment using microorganisms, Researcher in ecosystem restoration via site specific microbes. As we were concerned about public perception and stakeholder engagement of biotechnological solutions, we reached out to Tim Repas. He highlighted the importance of strong science communication. According to Tim, many promising ideas are rejected by the public eye due to the poor delivery of information or due to the presenter’s lack of understanding of their own proposal. In addition, he provided us with a method to confirm whether or not our system worked. He suggested 16s and 18s RNA analysis, which would be able to also detect any potential unwanted effects, such as an increase or decrease in the populations of other organisms. This is something we would employ when testing in a mesocosm or experimental lakes area.

Dr. Greg Pyle, University of Lethbridge: Follow Up Interview

We conducted a second interview with Dr. Pyle after we decided to utilize a CRISPR-Cas13a system. He appreciated that this solution would be more target specific, but also advised us of the stigma surrounding genetic engineering technologies. From looking further into this issue, we have learned that the way we communicate our solution to the public is key.

Dr. Jonathan Challis, Government of Canada: Research Scientist, Pesticide Chemistry & Ecotoxicology Dr. Challis’ research on Pesticide was very helpful in terms of how we would go about with our delivery system to our waterways. Our team also wanted to learn about governmental regulations that are placed on pesticides and their usage. As an environmental chemist and toxicologist who studies the state and behavior of contaminants in the environment and the impacts of these contaminants on organisms in the environment, he discussed in detail the importance in improving our understanding of when best to apply pesticides and in turn, our project’s delivery system. He highlighted the difficulty in mitigating the unforeseen effects of pesticide due to the nature of agriculture and the fact that it is directly implemented to the environment. As our project would also be directly set to an ecosystem, Dr. Challis noted that it takes numerous trials to make adjustments and create more specific and better application techniques.

Dr. Jim Davies, Innotech Alberta: Research Scientist in Microbiology, Mammalian Physiology, Ecological Models Dr. Jim Davies, gave very beneficial suggestions to propel our project to the next steps. We discussed at length what an actionable implementation of our project would look like in terms of getting approval on a federal and environment level. Our main area of discussion was in regard to the use of a mesocosm facility where our project could produce quantifiable results in the real world. Dr. Davies was also responsible for running mesocosm facilities in Vegreville, Alberta. He mentioned that they were seeking tenants for the 2024 season, and provided us with the criteria that we would need to meet to do so. According to Dr. Davies we must ensure that the cyanobacteria can be grown predictably. Specifically, where we intend to grow them and the time at which they begin to produce microcystins. Lastly, we talked through having a cereal-delusion in order to set a minimum concentration needed to achieve our desired reduction. This information was taken into account by our wet lab team.

Integrated Human Practices

From the start of our project we sought the input of experts and stakeholders on all aspects of our project and team. With the careful implementation of their suggestions, we were able to advance our project to its current stage. Prior to even creating our system we sought advice on organizing our team and how to come up with ideas effectively. From there, we had initially planned to deliver antimicrobial peptides to cyanobacteria. However, after hearing concerns in interviews about the lack of target specificity of such a system, we switched to a CRISPR-Cas13a system. This system allows us to target a gene that is unique to Microcystis aeruginosa cyanobacteria. Our wet lab team has performed various tests that have been recommended to us. Many of these tests are prerequisites to testing in mesocosm and experimental lakes facilities: a goal our team hopes to achieve in the future. This will provide an imperative expansion to regular laboratory bench experiments, which are limited in their ability to simulate aquatic environmental conditions. Finally, molecular modelling will allow us to optimize our system’s design, as well as improve both binding affinity and specificity. The input of experts has permitted us to optimize details and take the required steps to bring the project beyond the wet lab.

Entrepreneurship

In September of last year many of our team members had the wonderful opportunity to take part in an independent study-like course that is being run by Agility, a campus-wide program that is 100% donor-funded. Agility supports students of all disciplines interested in entrepreneurship and innovation, and provides instruction and tools to students who wish to learn more about those topics. The manager of this program, Brandy Old, is dedicated to coaching student entrepreneurs and encouraging the thought processes and mindset that will help future entrepreneurs to thrive in the modern market. As the majority of our team members are science majors, it was helpful for us to gain insight into entrepreneurship. Furthermore, it was interesting to learn how science and marketing can come together. The team completed this course after the 2021 iGEM season had finished, and shortly before the commencement of the 2022 iGEM season. One of the skills in particular, market discovery, was of great help this year. We implemented such strategies when looking for funding opportunities, attracting sponsors, and trying to enter collaborations with other organizations and companies.

Customers/Real World Implications

We have determined three possible demographics that would be interesting in the use of our system; government, agriculture, and water activity enthusiasts. As federal and provincial governments heavily regulate treatment applied to bodies of water, this demographic will play a big role. To discuss what steps we would need to take and what these policies may look like, we plan to get into contact with individuals involved in regulation and policy making regarding synthetic biology applications to bodies of water. The second demographic we have identified includes the agriculture sector. Albertan farmers often rely on man-made bodies of water that can be prone to cyanobacteria blooms, such as farm dugouts. While there are more treatment options applicable to man-made bodies of water, they still have their limitations. For example, many farmers choose to raise fish in their dugouts or have animals such as cattle that have access to the water. Chemicals such as copper sulfate treatments are toxic to beneficial aquatic organisms as well as animals. However, because of these off-target effects, many products using copper sulfate are no longer used or legally sold in Canada. The third demographic that may be interested in our proposed solution is the general public. Various recreational aquatic activities are interrupted each summer when cyanobacteria blooms form. Some examples of activities interrupted by cyanobacteria blooms include: Fishing: toxins can persist in fish from lakes with cyanobacteria blooms Swimming and other water sports,Boating and Pet owners.