Team:Lambert GA/Human Practices

INTEGRATED HUMAN PRACTICES

OVERVIEW

Lambert iGEM’s 2020 project, AgroSENSE, was inspired by a variety of experts in the hydroponics/aquaponics field. Hearing about timely, expensive, and often inaccurate methods of detecting nutrient levels faced by aquaponics farmers, the team had a goal of characterizing and improving upon a set of nutrient biosensors - specifically phosphate, nitrate, and nitrite. Lambert iGEM also utilized modular hardware to monitor environmental conditions for hydroponics systems. Furthermore, the team improved a previous frugal fluorometry device and app, FluoroCents, to create Fluoro-Q, another frugal fluorometer based upon image analysis. Hydroponics has the potential to provide high nutrient foods in urban areas which address several sustainable development goals of zero hunger and sustainable cities and communities.

In 2020 we were only able to complete a portion of these goals and the roots of our 2021 project were set. With the goal of improving the characterization of the phosphate biosensor and implementing the nitrate biosensor into a cell-free system, Lambert iGEM decided to continue AgroSENSE for the 2021 competition year. We further explored the safety issues associated with the distribution of biosensors, as well as the issues of plant diseases that diminish crop yields. The continued issues of accessibility of science and scientific tools led us to develop several frugal devices that were integral in our project as well as tools we can share with the larger community.

2021 Competitive Goals:

  • Improve characterization of the phosphate biosensor (BBa_K2447000).
  • Implement the nitrate biosensor (BBa_K372521 and BBa_K372522) into a cell-free system to address biosafety concerns.
  • Engineer plant pathogen biosensors for Fusarium (BBa_K3725080) and Phytophthora (BBa_K3725080) plant pathogens.
  • Engineer two frugal devices, a lyophilizer that eliminates safety concerns during transport and storage and a plate reader that quantifies optical density and fluorescence.
  • Collaborate with state level officials in the Department of Agriculture and House of Representatives to write regulatory guidelines for the use of biosensors in agricultural applications.

Last year, we developed biosensors to measure nutrient levels in the hope of increasing crop yields. This year, Lambert iGEM continued collaborating with stakeholders to increase the sustainability of hydroponics systems. We developed several creations to address the concerns and issues that arose from conversations with stakeholders. These issues included plant diseases that are prevalent in hydroponics, biosafety of E. coli-based biosensors for agricultural use, the costs associated with the implementation of biosensors, and the lack of public education. As our project progressed, we received feedback from various stakeholders and experts. We used this feedback to improve our project and address our sustainable development goals (See: Sustainable Development Goals).

Plant Diseases

During the 2020 season, we encountered a type of root rot in our hydroponics system. Unable to determine which of two common types it could be, Fusarium oxysporum or Phytophthora cryptogea, we contacted a local hydroponics farm, Sweetwater Urban Farms. Mr. Clint Crowe, the owner of Sweetwater Urban Farms, became an important contributor over the course of the year. During our first visit to his facility, we learned that plant pathogens were a major problem for him as a commercial grower as well as for the small-scale systems he sold to the public. Mr. Crowe cited Phytophthora cryptogea, a water mold that infects plants, as one of the diseases causing crop loss in his system. We also spoke with a Vegetable Disease Specialist from the University of Georgia, Dr. Bhabesh Dutta, about the lasting effects of the plant pathogen Fusarium oxysporum on Georgia’s agriculture. Dr. Dutta clarified that Fusarium oxysporum is highly prevalent in soil-based farms but also affects soil-less agriculture [1]. He informed us that the pathogen causes significant yield losses and economic consequences in the Southeastern United States [2]. He validated our efforts towards developing a plant pathogen toehold switch biosensor (See: PPB) to detect Fusarium oxysporum at an early state in hydroponics systems and soil-based farms. Proactive and early detection of diseases could allow farmers to diagnose and treat disease before it spreads, leading to improved crop yields. Agriculture contributes approximately $73.3 billion annually to Georgia's economy, according to the UGA Center for Agribusiness & Economic Development. Increasing crop yields could make a substantive improvement in our local economy and contribute to the United Nations Sustainable Development Goal of Zero Hunger.

Biosafety

One of our main goals for the 2021 season was to address the issue of biosafety; several potential stakeholders in our project, including Commissioner Gary Black from the Georgia Department of Agriculture and Mr. Clint Crowe from Sweetwater Urban Farms urged us to address the environmental risks of using bacterial biosensors in agriculture. To confront this, we implemented our nitrate biosensor into cell-free systems with the assistance of Dr. Adam Silverman from Northwestern University and Megan McSweeney from the Georgia Institute of Technology. Our phosphate biosensor could not be adapted into cell-free systems due to its use of membrane-bound proteins that are part of a signaling path native to E. coli, so we pursued alternate methods of ensuring safety. With the aid of Dr. Saad Bhamla from Georgia Tech, we developed a frugal lyophilizer (i.e. freeze-dryer) to preserve cells in an inactive state to mitigate biosafety issues in transport.

To investigate the feasibility of implementing our biosensors into the agricultural market, we contacted officials from the Georgia Department of Agriculture, specifically Commissioner Gary Black, Food Safety Director Natalie Adan, and Laboratory Director Dr. John Shugart. We learned that there are currently limited governmental barriers for biosensor distribution and disposal in Georgia. In response to this concern, we contacted Todd Jones from the Georgia House of Representatives and Ashley Haltom, the Vice President of Government Affairs from Georgia Bio to develop a regulatory proposal regarding safe biosensor distribution. We learned about the potential economic and environmental effects of our regulation as well as how it would affect agriculturalists in Georgia. This understanding will improve our perspective on biosafety as we revise our proposal for biosensor regulation to serve the needs of our community.

Adam Silverman

Megan McSweeney

Dr. Saad Bhamla

Sweetwater

Mr. Clint Crowe was instrumental in the progress of our project this year. He allowed us to visit the Sweetwater facility multiple times where we were able to obtain water samples and observe the prevalence and impact of the root rot Phytophthora cryptogea in tower farms. Visiting a commercial facility gave us valuable insight into the economics, sustainability, and viability of urban hydroponic systems. Mr. Crowe also provided feedback to our implementation team on the practicality of performing molecular based assays in the field as well as numerical data for our disease predictive model.

In April, Lambert iGEM reached out to Mr. Clint Crowe, owner of Sweetwater Urban Farms, to further understand common issues commercial farmers face while maintaining tower systems. Although he uses aeroponics, his system is comparable to hydroponics as they are both vertical tower systems that utilize liquid nutrient solutions as opposed to soil. We learned that he struggled to contain root rot which we hypothesized as being caused by Phytophthora cryptogea, a common plant pathogen. If gone undetected for a prolonged period, these pathogens can contaminate surrounding waters, thus infecting every plant in the system and resulting in browning leaves, severe wilting, and eventually death (Figure 1).

Consequently, Mr. Crowe often faces decreasing crop yield, money, time, and resources. At a state level, Georgian farmers lose 10-15% of soil-based crops to root rot and fungal infections; At a national level in 2018, farmers lost over $6.7 million [1]. Mr. Crowe emphasized the importance of timely pathogen detection and encouraged further development of our Phytophthora and Fusarium pathogen biosensors.

Although Mr. Crowe supported the development of our biosensors, he expressed his concern about potential E. coli outbreaks due to farmers’ limited knowledge about handling bacterial products. As a response, we reached out to the Georgia Department of Agriculture to identify if there were governmental restrictions on biosensor distribution. We learned that these do not currently exist. We asked Mr. Crowe for his input in developing a novel regulation to closely monitor the implementation and disposal of biosensors. As a potential consumer, he responded that biosensor regulation would be beneficial because it would provide a regulatory framework to implement in a variety of agriculture settings. We took in his feedback to ensure that our whole cell biosensors would only be utilized in an isolated chamber separate from the tower system.

We wondered if we could create a model that could predict the spread of Fusarium oxysporum, a fungus which spreads through airborne transmission of spores. Mr. Crowe generously shared specifics of his tower systems and farm layout to help us build our epidemiology model (See: Model).

Clint Crowe

Georgia Department of Agriculture

Lambert iGEM met with various members of the Georgia Department of Agriculture to understand the practicality of our biosensors from the government’s perspective. We first reached out to the Georgia Department of Agriculture, Commissioner Gary Black. Previously, we believed that the future of agriculture would shift towards hydroponics systems in urban communities due to their efficiency. Commissioner Black noted that while urban areas may be well suited for modular forms of agriculture, rural Georgia continues to rely on traditional agriculture as their primary export. He encouraged us to broaden the use of our biosensors to accommodate both modular and traditional forms of agriculture. This discussion prompted us to reach out to plant pathologists to eventually adapt our biosensors towards soil-based systems in order to improve the versatility of our project.

A discussion with Commissioner Black about legal protocols regarding the distribution of our biosensors revealed a gap in legislation regarding bioengineered products. He directed us to discuss regulatory issues with Georgia State Food Safety and Laboratory Directors, Natalie Adan, and John Shugart, respectively. After our conversations, we learned that since bioengineered products, in specific biosensors, are a recent innovation, there are limited legislative barriers and containment protocols regarding the safe distribution of our E. coli biosensors. As a team that works closely with E. coli, we understand the potential hazard bioengineered products pose to both farmers and consumers if handled incorrectly, prompting us to work with a legal advice team to develop a regulation regarding the distribution and legislation for the use of non-pathogenic recombinant biosensors in agricultural settings.

To approach this initiative, we contacted Ms. Ashley Haltom, Vice President of Government Affairs at Georgia Bio, to develop a timeline and immediate action items. She advised us to spend time researching the different perspectives of farmers about the implementation of biosensors on their farms to understand what concerns to address in legislation. She informed us about the timeline for the legislative process and encouraged us to reach out to Chairman Todd Jones, a member of the Georgia House of Representatives, for more information. At our initial meeting with him, we introduced our project and discussed a timeline to develop our legislation. Chairman Jones suggested that we shift our focus from legislation to regulation for a variety of reasons. First, the process of passing legislation is time-consuming, with the minimum duration being six months. In addition, since there will likely be future developments in biosensor research, amendments to legislation would be a similarly lengthy process. A pivot from legislation to regulation ensures that the governance of biosensors could be updated as often as necessary.

We are currently in the process of developing regulatory advice to share with the Georgia Department of Agriculture and the State Food Safety and Laboratory directors. We anticipate unveiling a draft in collaboration with our government partners early in 2022.

Commissioner Gary Black

Natalie Adan

Ashley Haltom

Chairman Todd Jones

REGULATIONS

Lambert iGEM emphasized biosafety throughout the development of our proposed implementation. After designing our nutrient and plant pathogen biosensors, we addressed potential biosafety hazards by developing cell-free systems, thus eliminating the risk of releasing bacteria to the public. Additionally, in order to transport our biosensor cells and lysates to the public in a freeze-dried, or a completely deactivated state, we developed our frugal lyophilizer, LyphoX. We collaborated with stakeholders in academia, industry, and government to determine the optimal disposal methods and materials, including but not limited to UV lights, ethanol, and bleach. We aimed to explore how the different aspects of our biosensor implementation tie into governmental policies.

Lambert iGEM met with various members of the Georgia Department of Agriculture to understand the practicality of our biosensors from the government’s perspective. In an attempt to determine if there were any legislative barriers hindering us from distributing our biosensors commercially, we reached out to the Commissioner of the Georgia Department of Agriculture, Gary Black. A discussion with Commissioner Black led us to discover a gap in legislation regarding bioengineered products: regulatory parameters for genetically modified E. coli did not exist on the state-level. To gather more information, he directed us to his Food Safety and Laboratory Directors, Natalie Adan and John Shugart, respectively. Ms. Adan echoed her claims, stating that in the event of an E. colioutbreak, there are limited standardized protocols to eliminate the bacteria, leaving it up to the farmers to choose a method they deem best fit for their farm. As a team that works closely with E. coli, we understand the potential hazard bioengineered products pose to both farmers and consumers if handled incorrectly. Although our biosensors are nonpathogenic and present in chambers separated from actual produce, they pose a risk if mishandled. As bioengineered products are emerging within industry, we believe it is crucial to proactively address potential concerns instead of waiting for outbreaks to happen. This prompted us to work with a legal advisement team to develop legislation regarding the distribution and regulation of non-pathogenic recombinant biosensors into the agricultural market.

To commence this initiative, we contacted Ms. Ashley Haltom, the Vice President of Government Affairs at the Georgia BioEd Institute, to create a timeline and a list of action items for our legislation. She advised us to spend time researching the different opinions farmers had about the implementation of biosensors in their farms, including potential economic losses that may arise with legislative barriers. She also informed us about the timeline for the legislative process and encouraged us to reach out to our district’s representative, Chairman Todd Jones, a member of the Georgia House of Representatives, to present our proposal.

In our first meeting with the representative, we introduced our project and idea for biosafety legislation. We discussed the extensive timeline for developing, revising, introducing, discussing, and passing legislation. Upon consideration of our goals and timeline, Chairman Jones suggested that we shift our focus from drafting legislation to a regulation. He stated that the process of passing legislation is time-consuming, with the fastest creation time ranging from six months to two years. In addition, since there will likely be future developments in biosensor research, amendments to legislation would be required to adapt accordingly, and would be a similarly lengthy process. A pivot from legislation to regulation ensures that the governance of biosensors could be updated as often as necessary. Considering Mr. Jones' perspective, we returned to the Georgia BioEd Institute to restructure our plan.

Our next steps center around gathering feedback from the Atlanta Farmers Coalition, a networking group of over 30 farms from across the state, to determine potential concerns and factors to include in our regulation. We intend to use their responses to work with a legal advice team to draft a proposal of a regulation for the distribution of nonpathogenic recombinant biosensors to be introduced to the Georgia Department of Agriculture and in the Georgia House of Representatives.

Georgia Bio

In February, we reached out to Georgia BioEd Institute, a division of Georgia Bio. Georgia Bio is a non-profit organization based in Georgia that promotes the interest and growth of the life sciences industry. Georgia BioEd Institute specifically works to strengthen life sciences education with initiatives that correspond with current industry needs. We communicated with Kristen Boscan and Megan Heaphy, who are the Director and Program Coordinator of the Rural Teacher training Initiative. Their initial feedback was to make sure our communications with communities were two way conversations and culturally sensitive in order to build long term relationships.

Later during the year, Kristin Boscan introduced us to Ashley Haltom, the Vice President of Government Affairs at Georgia Bio. During an October meeting with Ashley Haltom, we presented our proposal for a regulation that would address a need for safety in agriculture.

Kristin Boscan

Megan Heaphy

Dr. Dutta

In May, we contacted the University of Georgia’s Extension Vegetable Disease Specialist, Dr. Bhabesh Dutta, to discuss the lasting effects of the plant pathogen Fusarium oxysporum on Georgia’s soil agriculture. Dr. Dutta clarified that Fusarium oxysporum is more prevalent in soil-based farms and verified that the pathogen causes a significant economic detriment in southern Georgia with annual losses of “70 to 60% of fruit yield loss with wilted plants containing yellowed leaves.” [2] We used this information to add to our project (See: PPB) and consider a broader implementation of our Fusarium toehold switch biosensor.[3]

Dr. Bhabesh Dutta

JAN January 12, 2021 March 3, 2021 May 2, 2021 September 2, 2021 September 16, 2021 September 17, 2021 September 22, 2021 September 27, 2021 September 28, 2021 May 12, 2021 May 13, 2021 May 14, 2021 May 15, 2021 May 17, 2021 May 18, 2021 May 24, 2021 March 10, 2021 March 9, 2021 March 13, 2021 March 17, 2021 March 18, 2021 March 25, 2021 March 29, 2021 March 30, 2021 Adam Silverman from Northwestern University: Donated plasmids for NarL and NarX. Rural Teacher Training Initiative of Georgia BioEd Institute: Donated well combs made with our 3-D printer to enable rural teachers in Georgia to increase accessibility to hands-on molecular biology labs. Sweetwater Urban Farms:Visited their farm to learn about common problems they faced while maintaining their aeroponics towers, such as root rot and fungal disease. We also learned that nutrient fluctuations can increase susceptibility to disease. Lyopholizer call collaboration with MIT, JHU, UPENN, FSU, and UF Plate reader call with Dr. Bhamla, Chinna Devarapu, and Rajas Poorna Plate reader call with Rajas Poorna Lyopholizer call with Dr. Bhamla and Rajas Poorna Lyopholizer call collaboration with MIT, JHU, UPENN, FSU, and UF Yue Han, a graduate studentfrom the Styczynski Lab: Helped us troubleshoot issues we experienced with our nitrate biosensor model.Clint Crowe from Sweetwater Urban Farms: Explained that the potential negative impacts of biosensor regulation were limited, due to the minimal use of biosensors currently. Natalie Adan, John Shugart, and Brad Bush from Georgia Department of Agriculture: Learned there was a gap in regulation for the standardization of biosensors. Commissioner Gary Black from the Georgia Department of Agriculture: Spoke about the issue of plant disease as well as the potential feasibility of our biosensors. Dr. Bhabesh Dutta, a Vegetable Disease Specialist from the University of Georgia: Learned about the severe effects of Fusarium oxysporum on Georgia’s agriculture. Restricting Cloning Webinar Clint Crowe from Sweetwater Urban Farms: Learned that the early detection of root rot is important. Without early detection, he faces significant crop losses. He also spoke about his belief that hydroponics will be a long-term solution for food insecurity. Regulation’s first call with safety (Natalie Adan, John Shugart, John Bush, Susan Alexander) Lyophilizer call with Dr. Bhamla Sahana reaches out to Ms. Edwards and Mr. Martin about collaborating with them to put aquaponics kits in their sustainable agriculture classes Lyophilizer call with Dr. Bhamla from GT BhamlaLab Regina reaches out to Bonnie Plants Sweetwater Urban Farms: Experiences with plant disease.Megan Mcsweeeney from the Styczynski Lab: NUPACK software use which enabled us to develop the toehold switch biosensor sequences for plant pathogen detection. Zoya reaches out to Shelly Williams part of Georgia Ag Ed at Denmark HS and Mr. Dakota Martin part of FFA at North HS about if E.Coli in biosensors is a responsible, effective, and efficient method of addressing food insecurity Adam Silverman, Northwestern University: Helped us resolve complications we experienced during cell-free extractions. Neha reaches out to BioInnovate if we could have a brief presentation about our kits Commissioner Gary Black from the Georgia Department of Agriculture: Discussed the feasibility of implementing biosensors into Georgia’s agricultural market. MAR MAY SEP February 25, 2021 June 5, 2021 July 8, 2021 August 10, 2021 October 2, 2021 October 5, 2021 August 23, 2021 July 29, 2021 June 28, 2021 April 2, 2021 April 7, 2021 April 13, 2021 April 15, 2021 April 16, 2021 April 19, 2021 April 22, 2021 April 28, 2021 April 29, 2021 Megan Heaphy and Kristin Boscan from the Georgia BioEd Institute: Discussed the potential for science communication projects utilizing Foldscopes, frugal paper microscopes. Reached out to Kristin Boscan about how to work on Safety and Security Grant Sweetwater Urban Farms: Visited the farm to discuss the costs of hydroponics systems and what type of biosensor implementation would aid him best. Hardware received devices from Sweetwater Urban FarmsLyopholizer Call with Dr. Bhamla Sweetwater Urban Farms: Visited the farm to collect environmental water samples from his aeroponics system and help him plant garlic. Mr. Crowe, the owner, also talked about the increase in sales he experienced in the months of the COVID-19 pandemic. Lyopholizer call with Rajas PoornaPlate reader call with Chinna Devararpu Plate reader call with Dr. Bhamla and Chinna Devararpu Lyopholizer call with Dr. Bhamla Lyopholizer call with Dr. Bhamla Sheree Crowe from Sweetwater Urban Farms:Verified that their farm experienced root rot, and she invited us to their farm to speak about it in person. Plate Reader call with Dr. Bhamla Regina reaches out to Ugarden Horticulture DepartmentFred Conrad from Food Well Alliance emails back Lyophilizer call with Dr. BhamlaBonnie Plants Customer Service emails back - they have never experienced Phytophthora but they suggested we contact our local county extension educator (associated with UGA) Regina reaches out to Teena Atkins to ask some questions Teena Atkins from Farm Bureau forwards email to their county board division who are farmers. UGA Ugarden Horticulture Department replied saying they don’t have an aquaponic system an are just setting up hydroponic system and can’t answer any questions Neha reaches out to iGEM hq and put in collaboration request for the collaboration page on iGEM iGEM put out collaboration request on the page RUM-UPRM iGEM team reached out for our Restriction Digest FEB APR JUN JUL AUG OCT

REFERENCES

[1] Kratsch, H. & Omaye, S. (n.d.). Hydroponics: A Brief Guide to Growing Food Without Soil. https://extension.unr.edu/publication.
aspx?PubID=2756

[2] Banerjee, C. & Adenaeuer, L. (2014). Up, Up and Away! The Economics of Vertical Farming. Journal of Agricultural Studies, 2(1), 40-60. https://doi.org/10.5296/jas.v2i1.4526

[3] Wang, F. & Zhang, W. (2019). Synthetic biology: Recent progress, biosafety and biosecurity concerns, and possible solutions. Journal of Biosafety and Biosecurity, 1(1), 22-30. https://doi.org/10.1016/j.jobb.2018.12.003

[4] Suárez-Cáceres, G. P., et al. (2021). Susceptibility to water-borne plant diseases of hydroponic vs. aquaponics systems. Aquaculture, 544. https://doi.org/10.1016/j.
aquaculture.2021.737093

[5] Labconco. (n.d.). Freeze dryers. Labconco. Retrieved October 20, 2021, from https://www.labconco.com/category/freeze-dry-systems.

[6] Pauwels, E. (2013). Public Understanding of Synthetic Biology. BioScience, 63(2), 79-89. https://doi.org/10.1525/bio.2013.63.2.4

[7] Olson, H. A., et al. (2011). Phylogenetic History of Phytophthora cryptogea and P. drechsleri Isolates from Floriculture Crops in North Carolina Greenhouses. Phytopathology, 101(11), 1373-84. https://doi.org/10.1094/PHYTO-11-10-0302

[8] Blancard, D. (2012). Diagnosis of Parasitic and Nonparasitic Diseases. Tomato Diseases (Second Decision), 35-411. https://doi.org/10.1016/B978-0-12-387737-6.50002-9.

[9] Dutta, B. & Coolong, T. (2018). Fusarium Wilt of Watermelon in Georgia.
https://extension.uga.edu/publications/
detail.html?number=B1485&title=Fusarium Wilt of Watermelon in Georgia

[10] El Khoury, W. & Makkouk, K. (2010). Integrated Plant Disease Management In Developing Countries. Journal of Plant Pathology, 92(4), S35-S42. http://www.jstor.org/stable/41998886

[11] University of Georgia Center for Agribusiness and Economic Development. (2021). AgSnapshots 2021. https://caed.uga.edu/content/dam/caes-subsite/caed/publications/ag-snapshots/2021CAEDAgSnapshotsWeb.pdf

[12] Little, E. L. (2017). 2017 Georgia Plant Disease Loss Estimates. https://secure.caes.uga.edu/extension/
publications/files/pdf/AP 102-10_1.PDF

[13] Dremann, S. (2016). A plant killer with huge economic impact. https://paloaltoonline.com/news/
2016/09/23/a-plant-killer-with-huge-economic-impact.

[14] Snyder, W. C., & Hansen, H. N. (1940). The Species Concept in Fusarium. American Journal of Botany, 27(2), 64–67. https://doi.org/10.2307/2436688

[15] Georgia General Assembly. (2021). Tracking a Bill Through The General Assembly. https://www.legis.ga.gov/legislation/about

[16] Georgia Bio. (n.d.) About Georgia Bio. https://gabio.org/about/

[17] Georgia Bio. (2021). Strengthening Georgia's Bioscience Workforce Pipeline. https://gabio.org/georgia-bioed/