Dr. Anand Rao

Why We Talked to Them

We contacted scientists with expertise in Rhizobia and who are working on engineering plants to gather feedback on the scientific engineering design process and improve our engineering model of uptaking phosphate in Rhizobacteria. From learning more about selecting a plant to work with to the mechanisms of phosphate mobilization, we wanted to ensure that our project idea is a valuable contribution and a worthy use of Rhizobium.

Summary

Dr. Anand Rao provided advice on the genes we were testing, which helped us understand how they might cause other side effects and was also given special consideration during later trials. We learned that if we want to check whether a plant and microbe associate and a mutual symbiosis results, as inferred via nodule formation, we need to ensure a few key things for such a "nodulation assay" to be successful: the medium will not just barely allow, but truly support the growth of the microbial and plant partner in this symbiosis.

Our Takeaways

Dr. Anand Rao was one of the first people we contacted on our journey. Talking with him helped us better understand the full extent of our experimentation and the intricacies of how to grow our plants. Dr. Rao helped us decide what species of plants would be good to experiment on, and he gave valuable advice on how to keep them healthy and experimentally sound. We have taken into consideration Dr. Rao’s suggestions during our experiments and when analyzing the nodules that rhizobia can form.

Professor Uta Paszkowski

Why We Talked to Then

Professor Uta Paszkowski is a researcher at Cambridge studying mutually beneficial mycorrhizal (AM) symbiosis between plant roots and fungi. She has a wealth of knowledge on phosphate signaling and interactions between plants and bacteria/fungi. It was essential for us to gain the perspective of someone who understands so deeply the problem that we are trying to solve and our proposed solution. She explained that the main issue facing plants is their lack of ability to uptake phosphate. She also sheds light on the chemical properties of phosphate, which make it incredibly immobile in the soil, and how various conditions, like acidity, can change the way that plants uptake phosphate.

Summary

When asking us about our choice to use Rhizobium, she verified that rhizobium interacts with legumes, which are species-rich and an important group of crop species to consider. Through plants association with rhizobia, they also capture a large amount of nitrogen in the soil, which is important for plants to use at a later time. Professor Paszkowski also introduced us to mycorrhizal fungi, which naturally deliver phosphate to plants in an effective manner. As to our experimental design, she recommended that we work with a more compliant bacteria than Rhizobium to prove our concept and to find a true control for our plant experiments. Finding a valid control would ensure that we find a difference between what the rhizobium contributes to the plant and what the plant uptakes by itself, as well as if just the presence of the rhizobium increases uptake or if our genetic modification is responsible.

Our Takeaways

From our discussion with Professor Paszkowski, we took away a few different things. We shifted our focus to include not only phosphate pollution but also low phosphate availability. After being informed that the biggest issue was low phosphate availability and the phosphate being in a form unusable by plants, we chose to work on the symbiotic relationship between rhizobium and plants to assist plants in the uptake of phosphate. This was a change from our original idea of dealing with phosphate pollution by degrading it. In addition, upon hearing that we needed to find a true control, we worked on using arabidopsis as a negative control to function as a negative control for our plant experiments. Overall, our discussion with Professor Paszkowski helped us make our project more sustainable and contribute to overall responsible consumption by addressing the use of waning resources like soil phosphate.

Dr. Dave Jackson

Why We Talked to Them

Dr. David Jackson is a professor at Cold Spring Harbor Laboratory who studies plant regulatory genes and the way that they control plant growth and shape. In addition, he is a member of the community group Open Plant at Genspace, so he is well-versed in the structure, operations, and common pitfalls of community lab work. We wanted the specific viewpoint of someone already involved in community lab spaces and who understood plant signaling and plant synthetic biology. He was the first interview of our project and provided valuable direction in terms of the importance of our project and what challenges may be posed when it comes to implementing it. Also, he was also instrumental in helping us pick plants for our plant experiments and understand how that works.

Summary

He advised us to choose the plant that we want to work with based on growing times and how easy it is to work with. He suggested that we could utilize a legume as a model organism due to its popularity in the real world and its symbiotic relationship with Rhizobium, which has already been researched. In terms of implementation, he mentioned the regulations provided by the United States Department of Agriculture (USDA) for fieldwork. A key takeaway from this discussion point was that the USDA mostly provides regulations only for field work, not for lab work. To add on, Dr. Jackson spoke to us on the importance of repurposing inorganic phosphates from the soil - to reduce eutrophication caused by inorganic phosphate runoff and increase the level of phosphate usage, which is crucial as it is an incredibly limited resource. Dr. Jackson also mentioned that reusing phosphate within plants might be possible but can only be determined through research and experimentation. As a word of advice, Dr. Jackson informed us that science is about moving past roadblocks and having backup methods to ensure that there is still a way to progress on the project when something goes wrong.

Our Takeaways

Based on our advice regarding plant selection, we chose to use arabidopsis as a negative control since it does not form any root nodules and soy plants as a positive control (heeding his suggestion), along with various native Maryland plants for experimentation. Before our interview with Dr. Jackson, we were misinformed that arabidopsis produced root nodules, and our advice from him allowed us to change the design and implementation of our plant experiments to include it as a negative control. From Dr. Jackson’s comments, we chose to reach out to the USDA, hoping to glean information for our implementation plans. Additionally, we chose to simplify our project to focus more on researching the ability of Rhizobia to help plants uptake phosphate after hearing about the complexities involved with researching the repurposing of phosphate and recycling within the plant.

Dr. Reid Longley

Dr. Reid Longely is a microbiologist with expertise in root nodulation, specifically when it comes to nitrogen fixation.

Why We Talked to Them

We chose to reach out to Dr. Reid Longley because of his expertise in plant interactions with microbes. He introduced the plant microbiome and discussed plant-microbe interactions in the roots, the role of Rhizobia, and methods for interrogating the plant microbiome. Also, he explained that plants interact with diverse microbes, primarily bacteria and fungi. All plant parts are colonized by microbes both on surfaces (epiphytes) and inside tissues (endophytes). Additionally, he clarified that rhizobia’s main role is to fix atmospheric nitrogen into forms usable by the plant; however, they can only interact with limited plants, including legumes.

Summary

One of the key points of our discussion with Dr. Longley is that he introduced us to Pivot Bio, a company that is working on nitrogen fixation being engineered into corn, which the implementation subteam reached out to. Additionally, he emphasized the importance of considering that your engineered strain can compete with the microbes and survive in the natural environment while not breaking the other microbe interactions. Furthermore, he helped us understand the steps needed to take our project from the lab to implementation in the field and the role of math modeling. He suggested we conduct testing in a more natural environment. For example, the first test would be a single engineered stain in a plant. Then we can move it into a more native soil environment while still not being in the field (microbial population in a greenhouse). This information was useful in planning our implementation timeline.

Our Takeaways

We implemented Dr. Longley’s suggestions into our project by considering his advice in our experimental design. We chose to conduct our plant experiments in the greenhouse at the Baltimore Underground Science Space (BUGSS), to control the environment better. Additionally, his connection to Pivot Bio allowed us to reach out to them to gain information on the implementation side of our project. His suggestions also assisted us with our model. We used his advice on predicting the competition of microbes and their growth to create our growth curves of Rhizobia, adjusting for different variables.

Dr. Sharon Long

Dr. Sharon Long is a plant biologist who is well known for her pioneering research in plant genetic engineering, specifically with rhizobium nodulation and enhancing the uptake of nitrogen.

Why We Talked to Her

We wanted to talk to Dr. Long because we believed she would have special insight in our project since she has not only worked with plant microbiomes, but has expertise relating to our chassis of choice, rhizobium. Though she has not worked with phosphate, we believed that her work in genetically engineering to increase the uptake of nitrogen would help us as we worked on genetically engineering rhizobia to uptake phosphate.

Our Takeaways

Dr. Long gave us a lot of insight in regards to our experimental design for our plant experiments. She told us that the optimal temperature for the plants she experiments on is 30 degrees celsius, so we changed our plant experiments from an area 20 degrees celsius to an incubator that was 30 degrees celsius. She also encouraged us to record the pH of the soil we were growing plants in since that is a factor known to affect nodulation. We adjusted our protocol to accomodate this. Dr. Long also gave us guidance as to what kind of nutrients to have available for our plants in the soil we were using. We learned that we had to make sure that nutrients like potassium, phosphorus, and minor salts were available, but to keep the nitrogen concentration of the soil low so that plants would devote energy to nodulation.

Doug Myers and Madeleine Beller, Chesapeake Bay Foundation

We had the opportunity to discuss our work with the Chesapeake Bay Foundation, a non-profit dedicated to preserving the Bay and educating our community about sustainable practices.

Why We Talked to Them

The Chesapeake Bay Foundation is a prominent organization that works to educate, restore, litigate, and advocate for the Chesapeake Bay to ensure that it is possible to have clean water not only for our generation but for those following. We chose to reach out because we wanted the viewpoint of those involved in phosphorus pollution policy and to understand how this type of pollution has changed the bay. We were very fortunate to be able to speak to Dr. Doug Myers and Madeleine Beller. They helped us to understand the impacts of phosphate contamination, the sources of phosphate contamination, phosphate pollution reduction and monitoring, and the role of the public in reducing phosphate pollution. In addition, they provided us with feedback on our lab work, the implementation of our project, and the potential ethical/social concerns surrounding genetically modified organisms.

Summary

Dr. Myers expressed why phosphate is such a powerful pollutant - it’s sticky, causes an excess amount of phytoplankton, is released en masse after storms, and can be reused. Phosphate naturally comes from biological materials, like leaf litter, but it also comes from external sources, like animal manure fertilizers, which have been consistently overapplied, according to conventional agricultural practices. To add on, man-made structures, like drain tiles in fields and wastewater treatment plants, have increased stream erosion and contributed to pollution in the Bay. Currently, in Maryland, phosphate-containing laundry detergents are banned, and the phosphorus management tool is utilized to develop remediation plans, which have helped progress in reducing phosphate levels, as well as a series of statewide campaigns to increase awareness surrounding phosphate pollution. He also informed us that to monitor phosphate levels, scientists test in the field, in water next to the field, in receiving waterways, and in the Chesapeake Bay, using a test called the Melich-3 test. He instructed us to check out ambient monitoring set up by the state and USGS monitoring. As for the public’s role in phosphate pollution, the general public had to be informed of the detergent ban and the fee imposed on water bills to contribute to remediation. However, he also instructed us that there was still work to be done with education about urban construction and expressed that the CBF has been working on legislation to assist with stream restoration.

Our Takeaways

As to our own project, Dr. Myers and Madeleine Beller told us that we were on the right track, as phosphate needs to be uptaken by the plant but not made so soluble that it becomes an even more powerful pollutant. They suggested that we be mindful of the range of pHs, as phosphate solubility depends on pH. We integrated this advice into our project by including pH as a variable in our plant experiments and growing our plants in soil with varying pH. They helped us become aware of the fact that without considering pH as a variable, we could get false positives or negatives. Additionally, Dr. Myers mentioned that there would be fewer ethical concerns if we implemented our project in a closed system and that finding a way to target the impact of the Rhizobium better may be helpful. We integrated this advice into our project by changing our implementation concept to include no free, living Rhizobium and by consciously planning the placement of the bacteria to follow where the runoff is going based on topographical patterns. In her opinion, Ms. Beller outlined the best approach to starting a new educational campaign. She stated that listening to people and having meaningful, ongoing relationships with those affected was important. We used this advice for our education project, where we gathered the opinions of various teachers and community members before developing our educational materials. Overall, our discussion with CBF was very educational and triggered many changes to our project - both in experimental design and our calendar.

Jenna Schueller, Clagett Farm

To discuss how our project could contribute to sustainable resource use and consumption, we contacted Clagett Farm, a farm local to Baltimore that is dedicated to “farming practices that are truly sustainable both economically and environmentally.” They are practitioners of regenerative agriculture, agricultural practices that prioritize harmony with nature. This is done through nurturing and protecting soil health, biodiversity, and natural resources while also prioritizing economic sustainability and profitability.

Why We Talked to Them

We contacted farms to gather feedback from our proposed end users. We wanted to understand the challenges farmers face and ensure that our project will positively impact when implemented in the real world and address end users' concerns.

Clagett Farm is a farm that is committed to practicing sustainable and regenerative agriculture techniques for the betterment of the Chesapeake Bay and its watershed. We found it incredibly beneficial to gain the perspective of a farmer and someone involved in the management of the farm, who both understand the importance and practicality of sustainable agriculture, as an iGEM team that is developing a genetically modified organism to be used in conjunction with crop plants at farms and other locations. Speaking with experts at farms was helpful for us to understand the implementation of our project. Specifically, this discussion sheds light on the forms of innovative farming currently in use, how soil degradation and nutrient loss impact farms, the ways to test soil, and the balance between conventional and regenerative agricultural methods.

Summary

To start, Ms. Schueler explained what regenerative agriculture is - a sweep of practices combined in a farm to reduce pollution, increase soil health, and decrease emissions. One example of how Clagett Farms showcases this is through their intensive rotational grazing system and vegetable growing rotation. She then elaborated on the process that Clagett Farm has gone through to become a sustainable farm. Step by step, the farm recovered from years of use growing tobacco and corn exclusively. To monitor its progress, aggregate soil stability tests and soil core testing are performed. However, even with all of the scientific practices that the Clagett Farm employs, it is still a farm, which is a business, so each adjustment to the agricultural practices had to be economically sound. To encourage farmers to use more sustainable practices, certain state governments have set up programs that cover startup or installation costs for various practices. Regarding any social or ethical concerns surrounding genetically modified organisms (GMOs) in farms, Mr. Tana informed us that practically everything grown on farms has been genetically engineered to some extent, so there should not be many concerns with implementing a genetically modified product like ours. In addition, they reinforced the importance of our project due to the Clear Water Blueprint, which outlines a series of agricultural and wastewater reforms due to be completed by 2025 to reduce pollution in the Bay.

Our Takeaways

We took away numerous points from this interview, with the most crucial being the importance of profit. To make our project an attractive option for farmers, it must be both environmental and economical. We integrated this discussion point into our project by considering the economic implications of our project. Our implementation subteam researched a way to use our product that could help to improve yields or lower input costs, which is evident in our implementation plan. If this project were continued for another year, the economic benefits could be modeled, which would be consistent with the advice we received from Clagett’s representatives. This discussion encouraged us to think about how to help farmers and the plants, in addition to the environment.

Beth Kasulke, Hog Run Acres

Why We Talked to Them

Hog Run Acres is a small farm that supports the area around it. Ms. Kasulke, the owner, used to be a nutrient management officer, so she has a plethora of knowledge aimed towards allowing her plants to grow with the nutrients they need. We reached out to Hog Run Acres because we wanted the perspective of our end users, and the perspective of someone who is truly familiar with nutrient management. From this discussion, we were better able to understand how certain farms attempt to recycle as much as possible.

Summary

Ms. Kasulke reuses plant materials to feed her animals, thereby reducing the farm’s waste. When the plant waste material isn’t being used to feed animals, she also uses it for compost. This compost has to be tested, so the farmer knows what has been put into the soil, which will allow them to know what is being reused, and what is being lost. Testing beforehand allows farmers to know what nutrients need to be added to the soil for maximized crop output. The result of this is twofold - saving money for the farms and reducing wastage. Additionally, farms in the state of Maryland producing a certain crop yield or a certain amount of weight in animals have to have a nutrient management plan, which could be for example a buffer of grass around the edge of a field. The aim of this requirement was to reduce the amount of nutrient runoff from farms. Ms. Kasulke believes that this new take on plants, with projects similar to ours, could allow for many improvements and innovations on farms. She believes it could be put to good use in many ways, and perhaps even modified further to allow for additional benefits such as the modification of a plant to utilize the additional phosphate that is absorbed by our genetically modified bacteria. Such an improvement could then be utilized to allow crops to grow faster, causing more crop yields per season.

Our Takeaways

We gained multiple takeaways from this interview. Understanding the way that farmers focus on reutilizing nutrients puts our project into a wider perspective. For future work, it is definitely worth considering the possibility of engineering plants to interact with the excess absorbed phosphate. This would help to increase the sustainability of our project, as phosphate is a limited resource and multiple other stakeholders have stated that plants have issues uptaking immobile phosphates. In addition, the implementation subteam took into consideration the nutrient management plan brought up by Ms. Kasulke, and examined ways to integrate our project into existing nutrient management plans. The implementation subteam used the information provided by Ms. Kasulke to devise an implementation plan that explores the use of buffers, and other techniques that could be considered a part of a nutrient management plan. All in all, our discussion with Ms. Kasulke was very useful for our understanding of nutrient planning, reduction, and reuse in farms.

Shade Sabitu, Pivot Bio

Pivot Bio is a biotechnology company focused on producing microbial fertilizer that is more sustainable and safer for farms. Their product assists microbes to produce nitrogen for cereal crops, which eliminates the need for environmentally-harmful synthetic nitrogen. We chose to reach out to Pivot Bio because we wanted the corporate viewpoint of a project similar to ours. Our contact at Pivot Bio, Shade Sabitu, was incredibly knowledgeable on the regulatory processes required to manufacture and sell such a product.

Why We Talked to Them

We reached out to Pivot Bio because of their experience in working on designing bacteria in order to provide farmers with more efficient and sustainable nitrogen to fuel their crops. We wanted to ask for suggestions on how to implement our project in the real world responsibly. Their expertise in navigating the regulatory and approval process will be valuable as we work on applying our proposed solution to phosphate pollution.

Summary

Pivot introduced us to the concept of “Mode Of Action” (“MOA”), claims, and to marketing language. We were also told to identify our product classification - our project would be considered an inoculant. Furthermore, they suggested three potential agencies to which we could reach out to, the environmental protection agency (EPA), the United States Department of Agriculture (USDA), and state governments. Once we have identified the correct agency, the next step would be to begin the approval process, according to their requirements.

Our Takeaways

From our conversation we learned a lot about the various steps that would be needed to approve our project for use. Our implementation subteam took this information under consideration when planning the approval process for our project. Additionally, this discussion made our team mindful of the hoops that our project would have to go through, and a simplified statement of purpose for our project.

Deborah Hamilton, USDA

Why We Talked to Them

We decided to contact regulatory stakeholders to receive advice regarding the safety of implementing our project. Our goal was to address any ethical and safety concerns about our proposed solution to phosphate pollution.

Summary

After reaching out to the U.S. Department of Agriculture (USDA), Deborah Hamilton, who works on knowledge management for the USDA Food Safety Network, provided us with a Modified Microorganisms guide. The guide outlines the requirements and process for submitting permit applications for microorganisms developed using genetic engineering.

Our Takeaways

From this, we will consider the requirements when releasing our project in the environment. For example, we will think about the land area (size), how we will maintain the modified microbes at the release site and prevent their spread and persistence after the termination of a field trial, and how long and how often we will monitor to ensure modified microbes have not spread and will persist in the environment. We plan to continue our conversation with the USDA to better understand the regulations and how to implement our project safely.

Our Project’s Impact:

Phosphate is a limited resource. Soon, it may no longer be available, and right now, it’s not economically available because of fluctuating prices due to the Covid pandemic and the war in Ukraine. By using our project, we can reinvent the way we use fertilizer- not as a necessity, but as a safety net. We will be part of a new age for prevention of disaster for farms as a result of geopolitical distresses. We are part of a movement towards more sustainable, and even regenerative agriculture, which is a novel field in need of innovative technology. That is what we can provide.

By implementing our project with the procedure outlined above that was inspired by our talk with the Chesapeake Bay Foundation, we can mitigate the harmful effects of phosphate by making it available to plants. This is not just mitigating pollution, it is reusing a limited resource in a sustainable way.

Our proposed plan of implementation is to inoculate coastal plants in ditch areas where there are high amounts of fertilizer runoff by the Chesapeake Bay and Hudson River in an enclosed, contained bioreactor. The plants will then be harvested to be used as fertilizer by farmers.