iGEM 2020 — Team Gaston_Day_School

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# Team:Gaston Day School/Notebook

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[Safety](https://2020.igem.org/Team%3AGaston_Day_School/Safety)
[Human Practices](https://2020.igem.org/Team%3AGaston_Day_School/Human_Practices)
Awards

[Education](https://2020.igem.org/Team%3AGaston_Day_School/Education)
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# Notebook

Documentation of dry lab

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# Dry Lab Notebook Part 1

![Kudzu Background](https://static.igem.org/mediawiki/2020/0/01/T--Gaston_Day_School--Kudzu_Background.png)

#### Team

[Team](https://2020.igem.org/Team%3AGaston_Day_School/Team)
[Attributions](https://2020.igem.org/Team%3AGaston_Day_School/Attributions)
[Collaborations](https://2020.igem.org/Team%3AGaston_Day_School/Collaborations)
[Inclusion](https://2020.igem.org/Team%3AGaston_Day_School/Inclusion)

#### Project

[Contribution](https://2020.igem.org/Team%3AGaston_Day_School/Contribution)
[Implementation](https://2020.igem.org/Team%3AGaston_Day_School/Implementation)
[Description](https://2020.igem.org/Team%3AGaston_Day_School/Description)
[Design](https://2020.igem.org/Team%3AGaston_Day_School/Design)
[Engineering](https://2020.igem.org/Team%3AGaston_Day_School/Engineering)
[Notebook](https://2020.igem.org/Team%3AGaston_Day_School/Notebook)
[Results](https://2020.igem.org/Team%3AGaston_Day_School/Results)

#### Awards

[Education](https://2020.igem.org/Team%3AGaston_Day_School/Education)
[Model](https://2020.igem.org/Team%3AGaston_Day_School/Model)

[#### Parts](https://2020.igem.org/Team%3AGaston_Day_School/Parts)
[#### Safety](https://2020.igem.org/Team%3AGaston_Day_School/Safety)

[#### Human Practices](https://2020.igem.org/Team%3AGaston_Day_School/Human_Practices)

Email: igem@gastonday.org

Instagram: @gastondayigem

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# Team:Gaston Day School/Engineering

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Project

[Contribution](https://2020.igem.org/Team%3AGaston_Day_School/Contribution)
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[Description](https://2020.igem.org/Team%3AGaston_Day_School/Description)
[Design](https://2020.igem.org/Team%3AGaston_Day_School/Design)
[Engineering](https://2020.igem.org/Team%3AGaston_Day_School/Engineering)
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[Results](https://2020.igem.org/Team%3AGaston_Day_School/Results)

[Parts](https://2020.igem.org/Team%3AGaston_Day_School/Parts)
[Safety](https://2020.igem.org/Team%3AGaston_Day_School/Safety)
[Human Practices](https://2020.igem.org/Team%3AGaston_Day_School/Human_Practices)
Awards

[Education](https://2020.igem.org/Team%3AGaston_Day_School/Education)
[Model](https://2020.igem.org/Team%3AGaston_Day_School/Model)

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# Engineering

Engineering Success

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# Research

In order to solve the environmental problem that happens near us, our team chose to deal with the invasion of Kudzu in the United States, which will not only harm the normal agriculture of local plants but also will affect the other local properties, causing huge economic loss. In our original research, we thought that phytotoxin phaseolotoxin was only specific to kudzu plus other legumes and that the size of the target phaseolotoxin molecular is relatively large. But in the following discussion, our team found some concepts we used were wrong. As a result, we reread the related literature. In this process, we corrected some of our misunderstandings, found out the exact functions of each operon in the phaseolotoxin cluster (Aguilera, López-López, Nieto, Garcidueñas-Piña, Hernández-Guzmán, Hernández-Flores, Murillo& Alvarez-Morales, 2007), and gained a better understanding of the illicit transportation of phaseolotoxin in the bacterial Opp system.

# Imagine

In our project, our team expects to eliminate kudzu from the farmlands, power lines, and other places that have been invaded by kudzu in the United States. Many of the native plant species have been killed off due to their inability to compete with kudzu. The existing methods are mostly relatively useless for solving the problem permanently. This led us to the question of "How could we eradicate kudzu?". To tackle this question, we sought to use synthetic biology to come up with a method to control the growth of kudzu and reduce financial loss because of it.

# Design

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 [models](https://2020.igem.org/Team%3AGaston_Day_School/Model).

# Build

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).

# Test

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.

# Learn

After our first research, we learned that phaseolotoxin is not actually specific to kudzu, which would cause unexpected safety problems as well as difficulty in producing phaseolotoxin. The other thing is some *E.coli* strains are resistant to phaseolotoxin, and some are not, which will be an important point for us when we want to choose the *E.Coli* expression system in the future.

# Improve

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.

# References

Aguilera, S., López-López, K., Nieto, Y., Garcidueñas-Piña, R., Hernández-Guzmán, G., Hernández-Flores, J. L., Murillo, J., & Alvarez-Morales, A. (2007). Functional characterization of the gene cluster from Pseudomonas syringae pv. phaseolicola NPS3121 involved in synthesis of phaseolotoxin. Journal of bacteriology, 189(7), 2834–2843. https://doi.org/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

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

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

![Kudzu Background](https://static.igem.org/mediawiki/2020/0/01/T--Gaston_Day_School--Kudzu_Background.png)

#### Team

[Team](https://2020.igem.org/Team%3AGaston_Day_School/Team)
[Attributions](https://2020.igem.org/Team%3AGaston_Day_School/Attributions)
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[Inclusion](https://2020.igem.org/Team%3AGaston_Day_School/Inclusion)

#### Project

[Contribution](https://2020.igem.org/Team%3AGaston_Day_School/Contribution)
[Implementation](https://2020.igem.org/Team%3AGaston_Day_School/Implementation)
[Description](https://2020.igem.org/Team%3AGaston_Day_School/Description)
[Design](https://2020.igem.org/Team%3AGaston_Day_School/Design)
[Engineering](https://2020.igem.org/Team%3AGaston_Day_School/Engineering)
[Notebook](https://2020.igem.org/Team%3AGaston_Day_School/Notebook)
[Results](https://2020.igem.org/Team%3AGaston_Day_School/Results)

#### Awards

[Education](https://2020.igem.org/Team%3AGaston_Day_School/Education)
[Model](https://2020.igem.org/Team%3AGaston_Day_School/Model)

[#### Parts](https://2020.igem.org/Team%3AGaston_Day_School/Parts)
[#### Safety](https://2020.igem.org/Team%3AGaston_Day_School/Safety)

[#### Human Practices](https://2020.igem.org/Team%3AGaston_Day_School/Human_Practices)

Email: igem@gastonday.org

Instagram: @gastondayigem

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# Team:Gaston Day School/Experiments

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Awards

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# Experiments

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

![Kudzu Background](https://static.igem.org/mediawiki/2020/0/01/T--Gaston_Day_School--Kudzu_Background.png)

#### Team

[Team](https://2020.igem.org/Team%3AGaston_Day_School/Team)
[Attributions](https://2020.igem.org/Team%3AGaston_Day_School/Attributions)
[Collaborations](https://2020.igem.org/Team%3AGaston_Day_School/Collaborations)
[Inclusion](https://2020.igem.org/Team%3AGaston_Day_School/Inclusion)

#### Project

[Contribution](https://2020.igem.org/Team%3AGaston_Day_School/Contribution)
[Implementation](https://2020.igem.org/Team%3AGaston_Day_School/Implementation)
[Description](https://2020.igem.org/Team%3AGaston_Day_School/Description)
[Design](https://2020.igem.org/Team%3AGaston_Day_School/Design)
[Engineering](https://2020.igem.org/Team%3AGaston_Day_School/Engineering)
[Notebook](https://2020.igem.org/Team%3AGaston_Day_School/Notebook)
[Results](https://2020.igem.org/Team%3AGaston_Day_School/Results)

#### Awards

[Education](https://2020.igem.org/Team%3AGaston_Day_School/Education)
[Model](https://2020.igem.org/Team%3AGaston_Day_School/Model)

[#### Parts](https://2020.igem.org/Team%3AGaston_Day_School/Parts)
[#### Safety](https://2020.igem.org/Team%3AGaston_Day_School/Safety)

[#### Human Practices](https://2020.igem.org/Team%3AGaston_Day_School/Human_Practices)

Email: igem@gastonday.org

Instagram: @gastondayigem

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# Team:Gaston Day School/Results

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Awards

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# Results

An overview of what we discovered over the course of the year of working on our project.

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# Result

Despite not having the ability to work in the lab, we were able to find lots of information on kudzu and phaseolotoxin. Kudzu is an invasive species that came from Asia and has now consumed a lot of land in the southeastern states of the United States. There are many different methods of eradicating kudzu. These include the use of fire, mowing, chemicals, insects, goats, and fungus. These methods are all expensive, dangerous, or take many years. As a result of the difficulty to control kudzu, many plots of land go untouched because of Kudzu. In recent studies and modeling, our team discovered that in Gaston county, if kudzu was not managed it would cover over 300 hectares. For comparison, Gaston County is 94405.067 hectares. If this were to continue, we face problems as our population grows.

*Pseudomonas syringae* pv. Phaseolicola produces the chlorosis inducing toxin, phaseolotoxin. However, there are naturally occurring strains of these pathovars that do not produce this toxin and that does not even have the DNA that synthesizes it. Phaseolotoxin effectively inhibits the ornithine carbamoyltransferase (OTCase) activity in plants.

*Pseudomonas syringae* pv. Phaseolicola also produces a phaseolotoxin-resistant OCTase (ROCTase) to protect itself against its own toxin. ROCTase is a product of the arg-k gene and is only synthesized under conditions that lead to phaseolotoxin production. The argK gene is located in the chromosomal fragment in the Pht cluster, where genes involved in the production of phaseolotoxin are contained.

A quantitative microbiological assay for the phaseolotoxin was developed based on the ability of the toxin to inhibit the growth of *E.coli* on an arginine-deficient medium. Toxin-inhibition of these indicator strains can be shown to be OCTase to reverse growth inhibition. More specifically, phaseolotoxin is a reversible inhibitor of the enzyme ornithine carbamoyl-transferase, that catalyzes the formation of citrulline from ornithine and carbamoyl phosphate in the sixth step of the arginine biosynthetic pathway.

Moving forward with this information, we have a strong and supported hypothesis that the phaseolotoxin will target Kudzu’s fast-growing and regenerative system and reverse the growth inhibition. This process is projected to be as effective as using fire, however, it is more environmentally friendly because we can develop an off switch to protect surrounding life. After getting successful lab test results and proving that phaseolotoxin does have the ability to eliminate kudzu after experimental trials, approval from the local government and other related authorities are needed before we put it into commercial use. Additionally, the production of a large amount of our design may be another difficulty. We can try to make connections to universities nearby to find out if they will be able to improve our methods for volume production.

![Kudzu Background](https://static.igem.org/mediawiki/2020/0/01/T--Gaston_Day_School--Kudzu_Background.png)

#### Team

[Team](https://2020.igem.org/Team%3AGaston_Day_School/Team)
[Attributions](https://2020.igem.org/Team%3AGaston_Day_School/Attributions)
[Collaborations](https://2020.igem.org/Team%3AGaston_Day_School/Collaborations)
[Inclusion](https://2020.igem.org/Team%3AGaston_Day_School/Inclusion)

#### Project

[Contribution](https://2020.igem.org/Team%3AGaston_Day_School/Contribution)
[Implementation](https://2020.igem.org/Team%3AGaston_Day_School/Implementation)
[Description](https://2020.igem.org/Team%3AGaston_Day_School/Description)
[Design](https://2020.igem.org/Team%3AGaston_Day_School/Design)
[Engineering](https://2020.igem.org/Team%3AGaston_Day_School/Engineering)
[Notebook](https://2020.igem.org/Team%3AGaston_Day_School/Notebook)
[Results](https://2020.igem.org/Team%3AGaston_Day_School/Results)

#### Awards

[Education](https://2020.igem.org/Team%3AGaston_Day_School/Education)
[Model](https://2020.igem.org/Team%3AGaston_Day_School/Model)

[#### Parts](https://2020.igem.org/Team%3AGaston_Day_School/Parts)
[#### Safety](https://2020.igem.org/Team%3AGaston_Day_School/Safety)

[#### Human Practices](https://2020.igem.org/Team%3AGaston_Day_School/Human_Practices)

Email: igem@gastonday.org

Instagram: @gastondayigem

![Gaston Day Logo](https://static.igem.org/mediawiki/2020/f/fe/T--Gaston_Day_School--2020Logo.png)