Team:Gaston Day School/Poster

Poster: Gaston_Day_School

Gaston_Day_School Team Poster

Killing Kudzu: The Effect of Phytotoxin Phaseolotoxin on Kudzu Populations

Contributors include Brooke Dillingham, Kate Klinger, Kristine Huynh, George Luan, Neha Patel, Lydia Liu, and Matilda Zeigler.

Abstract

In order to remove kudzu, farmers and power companies often have to spray harmful chemicals on their land. These chemicals, along with many other kudzu removal options, are expensive and can pose harmful effects to human health and the ecosystem. Pseudomonas syringae pv. phaseolicola has been found to affect legumes specifically by producing the phytotoxin phaseolotoxin. Our team plans to replicate the phaseolotoxin production pathway in Escherichia coli and utilize the toxin as a more environmentally friendly option to remove kudzu. After the removal of kudzu, local endangered species can regrow without competition. Additionally, we developed three models to simulate the spread of infectious plant diseases like Asian Soybean Rust using kudzu as a vector, simulate the ROTCase production and activity under promoters with different strengths, and predict the spread and management cost of kudzu in a local area.

Introduction

Background

• Kudzu, originally introduced to Southern US for reducing soil erosion, is now spreading rapidly, taking up a large area of land
• Figure 1: Predicted Area Covered by Kudzu After 50 Years in Gaston County
• Powerline companies have to spend $1.5 million to repair damages caused by kudzu in the Southern US
• Kudzu “Tornado” and Broken Power Lines
• Threatened endangered plants in North and South Carolina
  • Kudzu affects other plants growing by disrupting growth conditions
  • Kudzu contains the active isoflavones that caused the affected soil hard to grow other plants
  • https://www.pbs.org/newshour/health/what-you-need-to-know-about-a-popular-weed-killers-alleged-link-to-cancer

Why is completely removing kudzu difficult?

• Absence of natural enemies of Kudzu in North America
• Dangerousness
  • Chemical herbicides might be harmful to other plants remaining in the area
  • Chemicals stagnate in other esculent plants that might cause people to be poisoned by eating these affected plants.
• Expensive
  • The chemicals for removing kudzu is expensive
  • The kudzu removing program costs a lot of money
• A long processing time
  • Need about ten years to completely eradicate the kudzu roots
  • Goats need 2-4 years to eat all of the kudzu’s roots.
  • The kudzu bugs help eating kudzu, but they only have been tested in the lab, but it also will eat the crops that are important to people.
• Finding a solution:
  • The phytotoxin phaseolotoxin produced by P.syringae pv. phaseolicola affects legumes(kudzu)’s growing
  • Lower risks to human and animals
  • Non-specific herbicide and will be applied in the area largely covered by kudzu

Inspiration

The hometown of Gaston Day school iGEM is in Gastonia, North Carolina. Gastonia’s leading economic venture is manufacturing with Jobs in this line of work employing the majority of citizens in the town (Sperling, 2019). However, there are many complications with manufacturing factories including harmful effects on the environment such as the emission of greenhouse gases causing water, soil, and air pollution and further contributing to climate change. This is the main concern with factories to the Gaston Day School iGEM team. Gastonia’s manufacturing economy puts high stress on our hometown’s environment, and these stresses are only further exasperated in the presence of the invasive species of Kudzu (How Can Factories Affect the Environment?, 2018).

North Carolina provides an optimal breeding ground for Kudzu, and as a result, the Kudzu population continues to expand throughout the state, including Gastonia. Kudzu consumes prime real-estate land as forests and free farming land shrink and where kudzu has no primal competition and where kudzu is resistant to pesticides on the market. The process to eradicate Kudzu from a site is long and tumultuous, often topping several years to remove a single root. It has pushed out countless local plant and animal species struggling to survive in their already industrialized community. Animals have to look for food sources elsewhere, seeing that no wild animals eat Kudzu (North Carolina Travel, 2015). Gaston Day iGEM plans to eradicate Kudzu out of a necessity to protect the plants, animals, and ecosystems of our community.

Problem

Pueraria montana, also known as kudzu, first came to the United States in 1876 from Japan. With no native predators to control it, kudzu rapidly overtook southeastern coastal states and has spread to cover more than 8 million acres of land. Oftentimes, those with kudzu on their lands are forced to use harmful chemicals in order to eradicate the kudzu. These chemicals, along with other kudzu removal options, are expensive as well as being harmful to crops, the environment, and humans. Phytotoxin Phaseolotoxin does not have severe dangers associated with fire, Roundup, or other means of eradicating Kudzu. Phytotoxin Phaseolotoxin is a safer alternative solution to other toxins since it does not pose severe harm to humans, animals, crops, or the environment. With the eradication of kudzu, native species will be able to reclaim their land, food, water, and other necessary resources to thrive again.

Idea

This year, our team’s goal is to eradicate kudzu from invaded regions through means of synthetic biology, specifically with the use of a safer and cleaner pesticide, Phytotoxin Phaseolotoxin. Phytotoxin Phaseolotoxin is produced by a strain of the bacterium called Pseudomonas syringae, and has been found to specifically affect legumes such as kudzu. Phytotoxin Phaseolotoxin can be cloned and expressed in E.coli. Our idea came from many interviews and observations from our team members and from professors at universities such as North Carolina State University.

Design

Figure 1: Urea Cycle and OTCase Pathway. Phaseolotoxin can be hydrolyzed to Psorn, an incredibly strong OTCase inhibitor that blocks the ornithine-citrulline conversion. Figure 2: Sequential Bi-Bi Mechanism of ROTCase. Our ROTCase Kinetics Model was built according to this mechanism.

Engineering

For the wet lab design, we are going to use the E. coli system expression to produce phaseolotoxin. Due to the pandemic, we were not able to access our lab. This forced us to change our focus toward creating models to find a way to apply phaseolotoxin in the real environment instead of our lab and to ensure that its side effect on the environment would be minimized. With the help of other teams, our team made some improvements to the model. The first model uses the cross-species transmission model from the University of North Carolina at Chapel Hill (Leer, 2005) (Fabiszewski, Umbanhowar, & Mitchell, 2010). The second model uses a Mass Action Kinetics as the model framework. NetLogo Model is mainly utilized for our third model (Aurambout & Endress, 2017). Apart from that, we could not make any validation of the experiment to produce phaseolotoxin due to the pandemic, so we ran our different models to explain the purpose of our project, optimize the E.coli expression system and predict the financial cost for the future product. The Pathogen Transmission Model and the ROTCase Kinetics Model are finished while the Kudzu Management Cost Model is still under testing.

Figure 1: Engineering Cycle for ArgK Expressions.

Figure 2: Engineering Cycle for Phaseolotoxin Productions.

Human Practices

This year, we sought to increase our community’s knowledge of synthetic biology, kudzu, our project, and iGEM. We created a Google Form where we asked people what their knowledge of synthetic biology, kudzu, and iGEM and if they had any questions relevant to the three subjects. After collecting back responses, we created a pamphlet that addresses the participants’ questions and put it on our Instagram account. In addition, we met with experts from the USDA and NC State, as well as the St. Andrews iGEM team to troubleshoot and get advice for our project. We learned a lot about modeling from St. Andrews in a series of meetings. From an expert from the USDA, we learned that after we eradicate kudzu from an area, the ecosystem can repopulate itself. We also got the idea to make a science education brochure because the expert from NC State stressed the importance of listening to the members of the community.

Room for Improvement

In the future, as long as the conditions permit, we will keep working on the wet lab to try to produce the phaseolotoxin in our lab. For the dry lab, the parameters of those models created need to be further optimized. If the phaseolotoxin can be made successfully, we still need to work on how to gain commercial approval from authorities and how to educate the potential users to use the phaseolotoxin correctly. What’s more, the ecosystem may be another part that our team should pay more attention to once the kudzu is removed.

Accomplishment

Bronze Medal

• Selected an imminent local ecological issue
• Created procedures and guides for future teams
• Added characterization information to J23100 and J23116

Silver Medal

• Designed a full engineering cycle and planning to complete once conditions permit
• Collaborated with three teams and attended three meetups in which we received and gave feedbacks
• Talked to experts and other iGEM teams to address concerns and usage of our project
• Proposed a safe and environmentally friendly plan of implementations

Gold Medal

• Addressed safety concerns and adjusted project goals according to expert advice
• Created three models to test our project and adjusted our designs according to the results
• Created a survey and a scientific brochure addressing the community concerns on synthetic biology and its implementation in our project
• Created a video on Western Blot and an iGEM themed bingo for the science community
• Promoted principles of diversity and inclusion on our team

Results

Positive Results with Success

Proposed end-users would be property owners or powerline companies that have large amounts of invasive kudzu on their land. With the eradication of kudzu, companies and property owners will be able to reclaim their land for use, which was once violated by Kudzu overgrowth. In addition, native species will be able to reclaim their land, food, water, and other necessary resources to thrive again.

Pandemic Setbacks And Lab Safety Measures

Due to the coronavirus pandemic, we were unable to safely perform lab work. However, once our team has the ability to get into the lab, we will try to use E. coli to produce phytotoxin phaseolotoxin under strict safety guidelines based on the model we created. E. coli is the only bacteria we have in our lab, so some safety aspects we need to consider is the risk of E. coli escaping the lab and then transferring/spreading its antibiotic resistance to other bacteria, possibly creating a large population of antibiotic resistant E. coli. The E. coli itself causes minimal harm in organisms, but it can cause irritation to the skin, eyes, respiratory system, and kidneys in humans (Felson, 2018).

Efficiency Of Phytotoxin Phaseolotoxin

We are also using Phytotoxin Phaseolotoxin, which is a non-specific plant toxin (Ferguson, & Johnston, 2008), so we can only apply it to areas with a high density of kudzu to prevent the toxin from killing other non-kudzu plants. The toxin is predicted to have an efficiency lower or equal to that of fire; however, it is significantly less detrimental to the ecosystem and more environmentally friendly.

Safety Guidelines for Phaseolotoxin

Phaseolotoxin is a plant toxin, so it is not very likely to cause harm to human health; in fact, it has the ability to control fast dividing cancer cells and might be a novel treatment for leukemia (Bachmann, Xu, Ratnapala, & Patil, 2004). However, extreme cases of prolonged exposure to Phytotoxin Phaseolotoxin might still affect humans’ ability to urinate and possibly cause hair loss. The phytotoxin phaseolotoxin will be used similarly as herbicides. Even though the toxin is much safer, the safety guidelines for using regular herbicides will apply. Therefore, we need to create a more detailed plan for both using E. coli and phaseolotoxin in our lab and make sure everyone who works on the experiment will be able to handle emergency situations.

Steps After Success (Or Precautions With Failure)

There are numerous substances to deactivate phaseolotoxin if the result is that the phaseolotoxin is not functional and has some other side effect to either the human health and the environment. However, with successful lab test’s results, approval from the local government and other related authorities are needed before we put it into commercial use.

Mass-Production and Future Steps

Large-scale production of our design may be difficult. We can try to make connections to nearby universities to see if they can improve our methods for volume production. At the same time, we will continue our human practice, which will be a crucial part of our future work. Our goal is to educate the local farmers about the increasing invasion of kudzu and its danger to the whole environment; furthermore, we will provide the farmers with some information about the biology of the phaseolotoxin and other concepts that appeared in our project.

References and Acknowledgements

Acknowledgements

The leaders of our team are George Luan, Kristine Huynh, and Anika Vercauteran.

The general members include Kate Klinger, Brooke Dillingham, Neha Patel, Yahoo Wu, Frankie Ni, Eric Lan, Lydia Liu, Gloria Zhang, and Matilda Zeigler.

Our team’s advisors are Anne Byford and Tina Heracklis.

We would also like to acknowledge of the supports we received during this project. Hereby, we express our gratitude to:

The Duncan Family for providing our team registration fee;

The Henry Family and The Hall Family for founding our lab;

Team Imperial College London 2020 for constructing the MATLAB code frame for our gene expression model and project mentorship;

Team St. Andrews 2020 for hosting the UK meetup, providing us the idea of the netlogo model, and troubleshooting our project;

Team TAU Israel 2020 for instructing us how to use SimBiology and Curvefitting toolboxes in great details;

Team William and Mary 2020 for hosting the Mid-Alantic Meetup.

Sponsors

References

Aguilera, S, Torre-Zavala, SD, Hernandez-Flores, JL, Murillo, J, Bravo, J, and Alvarez-Morales, A (2012). Expression of the Gene for Resistance to Phaseolotoxin (argK) Depends on the Activity of Genes phtABC in Pseudomonas syringae pv. phaseolicola. PLOSone 7(10):e46815

Aguilera, S, Lopez-Lopez, K, Nieto, Y, Garciduenas-Pina, R, Hernandez-Guzman, G, Hernandez-Florez, JL, Murillo, J, and Alvarez-Morales, A (2007). Functional Characterization of the Gene Cluster from Pseudomonas syringae pv. phaseolicola NPS3121 Involved in Synthesis of Phaseolotoxin. J Bacteriol. 189(7):2834-2843. Doi: 10.1128/JB.01845-06

Aurambout, J., & Endress, A. (2017). A model to simulate the spread and management cost of kudzu (Pueraria montana var. lobata) at landscape scale. Retrieved October 27, 2020, https://www.sciencedirect.com/science/article/abs/pii/S1574954117301097?via=ihub

Bachmann, P. D. A., Xu, R., Ratnapala, L., & Patil, S. (2004). Inhibitory effects of phaseolotoxin on proliferation of leukemia cells hl-60, k-562 and l1210 and pancreatic cells rin-m5f. Leukemia research, 28, 301–6. https://doi.org/10.1016/j.leukres.2003.07.002

Fabiszewski, A., Umbanhowar, J., & Mitchell, C. (2010, March 01). Modeling landscape‐scale pathogen spillover between domesticated and wild hosts: Asian soybean rust and kudzu. Retrieved October 27, 2020, from https://esajournals.onlinelibrary.wiley.com/doi/abs/ 10.1890/08-0820.1

Felson, S. (2018). E. Coli Bacteria Infection: Symptoms, Treatment, Causes & Prevention. Retrieved October 27, 2020, https://www.webmd.com/food-recipes/food-poisoning/ What-is-e-coli

Ferguson, A., & Johnston, J. (1980). Phaseolotoxin : Chlorosis, ornithine accumulation and inhibition of ornithine carbamoyltransferase in different plants. Physiological Plant Pathology, 16 (2), 269–275.https://doi.org/https://doi.org/10.1016/0048-4059(80)90041-7

Guertin, Patrick & Denight, Michael & Gebhart, Dick & Nelson, Linda. (2008). Invasive Species Biology, Control, and Research. Part 1: Kudzu (Pueraria montana). 24.

How Can Factories Affect The Environment? (2018). Retrieved October 27, 2020, from http://www.field.org.uk/how-can-factories-affect-the-environment/

Lambert iGEM Team. (2019). Hardware: Open Cell. Retrieved from https://2019.igem.org/Team:Lambert_GA/Hardware

Leer, S. (2005, April 25). Kudzu turning over new leaves in Indiana counties. Retrieved October 27, 2020, from https://www.purdue.edu/uns/html3month/2005/050425.Nice.kudzu.html

Newton, C., Nelson, L., Dewalt, S., Mikhailova, E., Post, C., Schlautman, M., Cox, S., Bridges, W., & Hall, K. (2008). Solarization for the control of Pueraria montana (kudzu). Weed Research, 48(5), 394–397. https://doi.org/10.1111/j.1365-3180.2008.00660.x

Rashid, M. H., Asaeda, T., & Uddin, M. N. (2010). Litter-mediated allelopathic effects of kudzu ( Pueraria montana) on Bidens pilosa and Lolium perenne and its persistence in soil. Weed Biology & Management, 10(1), 48–56. https://doi.org/10.1111/j.1445-6664.2010.00366.x

Sperling. (2019). Sperling's Best Places. Retrieved October 27, 2020, from https://www.bestplaces.net/economy/city/north_carolina/gastonia

The Invasion of Kudzu. (2015, July 08). Retrieved October 27, 2020, from https://www.northcarolinatravel.org/the-invasion-of-kudzu/