Team:Purdue/Integrated Practices

Purdue Biomakers




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Dow AgroSciences Trip

11 April 2016

A visit to Dow AgroSciences afforded us the opportunity to pitch our project to big industry early in the design phase, leading to an increased awareness of how our decisions affect future practicality and marketability. The ability to easily scale up a process is necessary for large-scale production, a consideration that led to the decision to encapsulate E. coli in xerogel beads, which can be rapidly produced.

Early in our project development we visited Dow AgroSciences’ Global Headquarters in Indianapolis. The purpose of the trip was to learn more about the agricultural industry, one of our target markets for implementation, and to receive input from scientists who work directly with agriculture regarding the feasibility and usefulness of our proposed project and prototypes. We attended a seminar on promoter design led by Dr. Hal Alper from UT Austin, had a round table discussion with Dr. Alper and Dr. Steve Evans of DAS, took a tour of DAS, and then presented our project to a group of DAS employees ranging from experts in the field, those with previous iGEM experience, businessmen, and experienced financial consultants.

Takeaways from the day included valuable advice on the practicality of our proposal, considerations for implementation, and excitement for our idea. While DAS does not manufacture biological solutions such as ours, the company does consider the importance of capturing phosphorus runoff to keep it from entering the water supply and approaches the problem in a variety of other ways. Naturally, a large emphasis was placed on the cost effectiveness of our solution; after all, in order for science to be accessible to the public, it must be competitive with other products on the market. If we want farmers to implement our method of phosphorus removal and energy generation, we must prove that it is favorable to them to do so. What would their energy savings be, and how would they benefit from collecting and reusing phosphorus runoff? What would their savings on fertilizer be? Questions like this had a direct impact on our project as we built our model, causing us to consider the comparative economic advantage of our solution as we completed cost analyses on alternative methods.

Dr. Jay Garland

15 April 2016

Dr. Jay Garland of the United States Environmental Protection Agency (EPA) discussed with us the sustainability and integration of our project. Based on our conversations, we more seriously pursued safety mechanisms and considered the role of our modified E. coli in microbial ecologies.

As soon as we identified tentative project ideas, we searched for policy expert contacts to help focus our ideas and guide our project design. After talking with one of our advisors, we contacted Dr. Jay Garland, an EPA scientist involved in green infrastructure development. Dr. Garland is a microbial ecologist who worked on bioregenerative systems at NASA for 20 years before working at the EPA where he has completed several projects involving water contamination and wastewater management.

We had a lovely conversation over the phone and stayed in contact with him throughout the summer. As he has history with NASA, we considered the potential space application of our systems.

During our first conference call discussing the mass balance of phosphorous in wastewater treatment and in runoff, Dr. Garland made sure that we were also focusing on the bigger picture and problems with phosphorus: the lack of phosphorous recovery in wastewater and the runoff from farmland that causes eutrophication. We also discussed current wastewater management and how these practices could be considered unsustainable (especially in regards to phosphorous removal) because of the heavy use of chemicals in the process. In viewing wastewater as a microbiome, how could we best engineer a synthetic ecology through the implementation of our technology? How would our solution fit into a global context in terms of its cost, energy usage, and environmental impact?

Bearing these questions in mind, we took the specific step of developing a theoretical prototype with solar panels that would facilitate net zero energy usage by the system. It was also important to keep the physical prototype as inexpensive as possible if it were ever to be used by farmers, wastewater treatment facilities, or developing countries. Thus, the five-gallon bucket system was born using materials that could be easily purchased from any hardware store. Contact with Dr. Garland encouraged a whole-system, global perspective that may have been lacking without his insight.

Trip to West Lafayette Water Treatment Facility

26 May 2016

Visits to the West Lafayette Wastewater Treatment facility enabled us to see wastewater treatment processes first-hand and ask employees valuable questions. Notably, West Lafayette made a cost-driven decision to implement a chemical method of phosphorus remediation with the intention of changing to a biological one in the future. In developing a viable solution, we heavily weighted low cost to maximize the number of application scenarios in which it could be effective.

In an effort to determine how our project might best impact our community, we reached out to Sara Peel, Director of Watershed Projects at the Wabash River Enhancement Corporation (WREC). She sent us several documents containing statistics, data, strategic planning, and policy regarding Indiana and the Wabash Rivershed area’s water usage and measurements. This information was used in analyzing sources of phosphorus input to the Wabash, the greatest of which was found to be agriculture.

Pursuing further information about the Lafayette community’s water management systems, we joined Peel and Angela Andrews, City of Lafayette Water Pollution Control Chief of Surveillance at the West Lafayette Wastewater Treatment Plant. In prior research, we found that both the Lafayette and West Lafayette municipalities operate combined sewer systems into which flow both runoff and building sewage lines. This leads to combined sewer overflow (CSO) during periods of heavy precipitation or springtime snowmelt, when water from streets is in excess, spilling sewage, nutrients, and sediment into the surrounding watershed. As the cities seeks to solve this issue, larger pipes are being installed; Peel and Andrews suggested that this may be a possible implementation point of our phosphorus removal technology.

One of the most interesting discoveries came midway through the conversation when we learned that the facility would implement a chemical method of phosphorus removal utilizing ferric chloride on August 1 of this year. This surprised us on two fronts. One, why didn’t a phosphorus removal system exist already? Two, why a chemical as opposed to a biological method? In response to the first question, we found that the facility had been newly permitted for phosphorus removal; as awareness of the issue of excess phosphorus spreads, this is becoming more common, but it is not yet standard practice. Regarding the second, a biological system requires a larger capital investment upfront. Because this is an election year, the chemical system is being used as a cheaper “bandaid” with the intent to switch to a biological approach long-term.

We gained insight into the tough balance between real-world budget, politics, and environment and many new ideas for applications. This conversation launched an investigation into at-the-source points of technological implementation such as in field tile drains, as Peel suggested, and swirl collectors in parking lots, mentioned by Andrews. Interest in field tile drains sparked the attendance of Dr. Andi Hodaj’s thesis.

USDA National Soil Erosion Research Lab

3 June 2016

The United States Department of Agriculture National Soil Erosion Research Laboratory was instrumental in guiding our project to completion, influencing many stages of design and application, specifically related to tile drain. From speaking with representatives of the USDA-NSERL, we learned more about the specific application of our phosphorus mechanism in scenarios useful for farmers; bearing this in mind, we ultimately designed our bioreactor prototype to be useful in controlling effluent from a tile drain.

As we continued to develop our implementation plan for our project, we took a tour of the USDA National Soil Erosion Research Lab to discuss tile drainage and biodigester plans. We toured the facility with Dr. Chi-Hua Huang and Dr. Ashley Hammac who have since attended all of our weekly lab meetings. Dr. Huang is a veteran of the NSERL and is considered a soil expert, while Dr. Hammac is a relatively new addition to NSERL and has started work at NSERL as an Agronomic Cropping Systems Conservationist.

Both have been instrumental throughout our project. They allowed us to use their analytics lab to test water soluble phosphorous levels, lent some of their anaerobic chambers for the growth of our Shewanella and engineered E. coli, and have continually provided insight on both the scope and the practical real-world applications of our project.

While on the tour of the facility we learned about other methods of phosphorous removal and discussed the benefits and problems of our solution. We learned that the NSERL scientists are currently exploring steel slag and limestone as methods of taking up phosphorus along ditches and in depressions in fields. Steel slag seems to be better at taking up and holding phosphorous; however, it does not release the phosphorous, and there are no current methods to induce this. From this discussion we decided that potential usage of accumulated phosphorus would be a benefit of our project in comparison to other strategies. Additionally, we identified the steel slag as another potential solution to phosphorous in water that we could use to compare to our final system.

We also discussed the potential problem of our engineered bacteria being out-competed in both wastewater treatment facilities and bioreactors by native species that are better adapted. This prompted us to consider how we might make our cells more viable and to research past iGEM projects that deal with cell viability.

A mechanical engineer, Mr. Scott McAfee, who works in the lab offered to help us build a bioreactor to test one of our application areas, and we began to design the physical structure to validate and test our mathematical model.

Dr. Andi Hodaj’s Thesis Defense

6 June 2016

Dr. Andi Hodaj’s thesis defense about tile drain modifications for improved phosphorus uptake enabled the team to consider competing or alternative mechanisms of phosphorus uptake and how our solution might enhance developing or developed systems. We increased the modularity of our system to improve its chances of seamless integration into existing systems.

Early in the development of ideas for the application of our system for phosphorus removal, we attended the thesis defense of Dr. Andi Hodaj, a Ph.D. student in Agricultural and Biological Engineering at Purdue University. Entitled “Exploring the potential of a two-stage ditch as a tool to reduce nutrient loads,” Dr. Hodaj’s thesis presented the multi-faceted hypothesis that two-stage ditches have increased impact on reducing sediment, total phosphorus, nitrogen, and soluble reactive phosphorus and represented the two-stage ditch in the hydrologic model as a conservation effort, simulating mechanisms of nutrient retention within the cycle.

A two-stage ditch is a specialized agricultural ditch that has banks dredged to create small floodplains upon which nutrient-absorbing vegetation is planted. The floodplains drastically increase the amount of time that water and vegetation interact in addition to dissipating the energy of high-flow water, maintaining necessary volume while reducing velocity. This strategy was shown to decrease the phosphorus load of agricultural runoff by approximately 40 percent. Dr. Hodaj cited a future application of his work as impacting the analysis and comparison of alternative methods such as dephosphorizing bioreactors. You can imagine the team perked up when he mentioned “dephosphorizing,” but we hadn’t yet thought of linking that technology to a bioreactor.

Dr. Hodaj’s defense further solidified our desire to pursue an agricultural application for phosphorus removal, ideally in a tile drain or ditch scenario. It also sparked further research into the possibility of housing our E. coli in a bioreactor that could be installed by technicians in wastewater treatment plants or farmers at their fields. Ultimately, this bioreactor became our prototype; thank you, Dr. Hodaj, for inspiring us!

Midwest Clean Energy Forum

9 June 2016

The Midwest Clean Energy Forum challenged us to focus on how we could make our technology effective for as many people as possible as quickly possible. Purdue University President Mitch Daniels said in an opening statement, “It’s only an innovation when it is useful to someone,” which drove decisions to further strengthen our relationship with the USDA-NSERL and actively seek opinions from experts in the agricultural sector.

With a more concrete idea for our project in place, our team then sought out resources for defining how we could practically implement our design. This led us to the Midwest Clean Energy Forum--an annual event hosted on Purdue’s campus where members of U.S. Congress, the U.S. Department of Energy (DOE), U.S. Department of Agriculture (USDA), and representatives in research and industry gather to discuss problems and pathways to a future of clean energy.

Although we attended with the intention of talking to the panelists covering the implications of synthetic biology in energy, what we quickly found was that some of the most interesting and thought-provoking questions and comments posed came not from those directly related to our field but from policy experts and industry leaders in other areas. To give an example, when first addressing his audience of academics, politicians, and business people, Purdue University President Mitch Daniels stated, “It’s only an innovation when it’s useful to someone.” The least exciting kind of discovery is the one that never leaves the lab bench, never escapes from the dark laboratory corner into which its dusty box is shoved, and never has the opportunity to make a positive impact on a single person. In that moment, we resolved to pursue the necessary steps to make our technology publicly accessible, construct a business model if possible, and by assuming a design mindset of practicality and feasibility.

Dr. Franklin Orr, the U.S. Under Secretary for Science and Energy and principal advisor to the Secretary and Deputy Secretary of Energy on clean energy technologies and science and energy research initiatives, was the Forum’s distinguished guest. His opening remarks cited humans as a global, biogeochemical force affecting all aspects of earth. “We need all the tools in our toolkit plus those that have yet to be invented,” which is what we, the inquiring minds of iGEM, hope to do--create tools to harness this unstoppable human force for good.

Eric Holcomb, Indiana Lieutenant Governor, and Jim Merrick, Indiana state senator, the next speakers to command the floor, both highlighted the importance of using diversified regional resources to sustain our country’s energy needs. This caused us to consider the practicality of our solution in the Midwest; fortunately, Indiana has no shortage of corn, soybeans, and other agricultural activity to generate enough phosphorus load in our water for our uptake method to be a relevant solution to a pressing issue.

The biggest lesson of the day was woven into many comments, with quotes like, “Cost really matters in these things,” “If it’s painless, people will participate,” and “If we build it, they may still not come,” written in the margins of our notes. All these statements are iterations of the idea that in order for any innovation to be relevant, let alone successful, it must first be accessible to the public. We further broke this down into three specifications: One, our solution must be cost-effective; two, our solution must be easily integrated into existing practices; and three, our solution must be approachable and intuitive.

Along this same line, Dr. Peter Keeling of Iowa State University specifically mentioned the importance of the integration of biology and chemicals. This raised an important question--how can we integrate our biology with chemicals already used in phosphorus treatment? We sought to resolve this issue by paying a visit to the West Lafayette Wastewater Treatment facility later in the summer.

Meeting with Dr. Thomas Sors

21 June 2016

Dr. Tommy Sors, Chief Liaison of Bindley Bioscience at Purdue University, met with us throughout the summer to discuss our progress and specifically helped us to narrow the scope of our initial project to present a clear and cohesive objective: phosphorus uptake, storage, and exportation.

As the team began wetlab work, we were approached by Dr. Thomas Sors, the Chief Liaison for our lab in Bindley Bioscience Center. He was especially interested in talking with us about our project and discussing ways in which he could help with lab management and faculty connections. We set up a meeting and, upon arriving, found Dr. Sors to be well-versed in our project. He asked several questions about the logistics of our characterization process and expressed some concerns over the scope of our project components.

We had tentative ideas in mind, but after talking with Dr. Sors we established a solid timeline for our characterization lab work. He also put us into contact with Dr. Larissa Avramova whose equipment we would need to use, and he helped set up trainings for various machines throughout Bindley that we would be using.

However, he was concerned that we were doing what amounted to two projects and said that we might want to focus on just one for the sake of both time management and cohesiveness of our Jamboree presentation. He did concede that if we could demonstrate enough of a connection between the two projects and felt we had time, then we could conceivably move forward with both.

This prompted us to seriously consider how we presented our project. We had originally imagined it as one project and wanted to make sure that it came across as such. The final prototype design work was continued, but we strove to better our explanation of the two components by stressing the similarity: water cleanup for the generation of clean drinking water.

Dr. Ron Semmel

29 June 2016

Through a meeting with Dr. Ron Semmel, a Monsanto scientist, the team acquired useful contacts and information regarding local phosphorus and nitrogen soil concentrations. Among other topics, we discussed phosphorus regulatory policy, specifically as it relates to agricultural usage, informing us of the current political discussions surrounding nutrient load.

Through a meeting with Dr. Ron Semmel, a Monsanto scientist, the team acquired useful contacts and information regarding local phosphorus and nitrogen soil concentrations. Dr. Semmel concisely described his job: “They send me things. I try to kill them. If I can’t kill them, they go to market.”

“Production Agriculture and Environmental Targets: Can They Coexist?”

7 July 2016

Dr. Kevin King helped to expand upon Dr. Semmel’s characterization of the phosphorus issue within agriculture by discussing the specific problem of phosphorus buildup in soil resulting from its tendency to “stick” to particles. Bearing this “stickiness” in mind, the we better-understood the need to facilitate understanding between farmers and policy-makers in creating fair regulation and implementing solutions.

Before beginning work one morning, Dr. Rickus, the team advisor, brought to our attention a seminar scheduled for that afternoon entitled “Production Agriculture and Environmental Targets: Can They Coexist?” presented by visiting Purdue graduate Dr. Kevin King

Dr. King is Research Agricultural Engineer with the USDA-Agricultural Research Service Soil Drainage Research Unit in Columbus, Ohio and 2016 ABE Outstanding Alumnus. He highlighted edge-of-field research aimed at quantifying the impacts of agricultural production practices and discussed potential management practices that might be used to reduce offsite nutrient transport and meet the established water quality targets.

Opening with the question, “Can we optimize production while minimizing environmental impact, bearing in mind that systems are leaky?” Dr. King gave a brief history of agriculture in the U.S. and the prominent use of fertilizers, defined “edge-of-field research,” and discussed the “legacy phosphorus issue” in detail. Tile drainage was identified as a significant pathway of phosphorus loss, leading the team to further investigate an at-the-source implementation of our phosphorus-munching microbes. To analyze the metrics of nutrient loads carried by these tile drains, surface runoff and drain discharge measurements are taken literally at the edges of fields, an automated sampling unit collecting data year-round. This data indicates that while water arriving at the termination of a tile drain contains less phosphorus than water traveling across the land’s surface, its phosphorus content is still above a critical level necessary to prevent unwanted algal growth.

A sustained, sometimes unnecessary application of phosphorus to fields has led to a build-up in the system known as the “legacy phosphorus issue.” Dr. King noted that while nitrogen is capable of passing through agricultural systems, phosphorus is not, due to its natural ability to bind itself to soil. Annual fertilization using mixtures containing phosphorus exacerbates this problem; Dr. King suggested that there is currently enough phosphorus to grow crops for an indeterminate time without additional application, which would save depleting minable reserves and reduce phosphorus in water.

In response to the question posed by the title of his presentation, Dr. King did not speak directly. However, one of his comments led the team to draw positive conclusions: at the end of a talk, farmers often approach him asking about measurement technology and specific steps they can take to reduce nutrient load coming from their fields, and they express desire to use their land to participate in his studies. This demonstrates that lack of awareness, not necessarily lack of desire, inhibits a positive relationship between seemingly dueling agendas. Through public education and outreach, production agriculture and environmental targets can not only coexist; together, they can thrive.

Wabash River Fest

9 July 2016

At the Wabash River Fest we discussed our project with the public and started a discussion around a synthetic solution to the phosphorus problem. The responses we received helped inform not only our design but also future communications and presentations as we realized the need for framing our solution through a discussion of the phosphorus problem with concrete examples.

As an outreach project, our iGEM team hosted a booth at the Wabash River Enhancement Corporation’s (WREC) annual Wabash River Fest. It was great fun for all parties involved as passersby learned about DNA, built (and ate) their own licorice and marshmallow double helix models, and asked questions about synthetic biology and the phosphorus cycle. Young scientists tried their hands at pipetting, mixing colored water in eppendorf tubes to create vibrant new hues. In the days leading up to the Fest, we prepared 150 build-your-own candy DNA kits, and we ran out just after lunch! The event was well-attended; we appreciated the opportunity to spread our passion for synthetic biology with young and old alike.

Memorable passersby included a first-grade boy with infinite intelligent questions whose grandfather told me in limited English that their family had immigrated from China only several months ago; a woman who spoke in-depth of her passion for biology and desire to become a research scientist; a World War II veteran who listened intently to our story, asked a couple questions, then related synthetic biology to his woodworking business; and a small girl from whom we had to extract the micropipette for other children to use, lest she spend the next hour pipetting to her heart’s content.

We also used this event as an opportunity to distribute a survey about wastewater treatment practices whose results you can find on our [Survey page link]. The population was varied and diverse, making the River Fest an ideal place to gather a random sample of data. Overall, the day was well-invested as the public learned more about the phosphorus problem and we learned the value of small moments connecting with others.

Weekly Lab Meetings

Throughout the summer

Advisory meetings with our sponsors were integral to our success, and their influence is pervasive throughout design and execution. For an hour every Wednesday during the summer, leading experts provided meaningful feedback on our progress over the previous week, often suggesting specific edits to protocols, sometimes re-directing our experimental focus, and always encouraging us to iterate our designs.

A Purdue University iGEM team tradition are weekly meetings open to the campus in which we present our recent accomplishments, goals for the upcoming week, and questions of any sort to our advising professors, Drs. Kari Clase, Jenna Rickus, and Kevin Solomon, Purdue student researchers in our building, and other visitors. These meetings have a tremendous impact on our work, as we often find ourselves evaluating our trajectory and making significant changes to experimental design by their conclusion.

For example, conversations with Dr. Solomon led to the revision of our genetic constructs at the beginning of the summer and again toward the end as we decided to use Gibson assembly to construct our phosphorus-uptake operons. Dr. Rickus guides us with engineering considerations, asking for data to back all of our decisions. This summer, Dr. Ashley Hammac of the U.S. Department of Agriculture National Soil Erosion Research Laboratory (USDA-NSERL) has been immensely helpful as he lends us his phosphorus expertise, pointing out, for example, that phosphorus is a “sticky” and we should take that into consideration when dealing with glassware that has the potential to leak phosphorus once “stuck” to it into any media it may contain. This specifically led to modifications to our phosphorus uptake analysis protocol.

Weekly lab meetings are hugely important in our work, and we would be at a loss without the assistance of the professors who make our project possible.