Team:Bielefeld-CeBiTec/Description

Abstract

Even though the past was not peaceful in terms of conflicts between several countries, we can live peacefully nowadays. Nevertheless the past still hides its danger in our environment and society. Due to the World Wars chemical weapons are still stored in storage sites, known as well as unknown. These chemical weapons were developed in the past during the World Wars because of research results in chemistry which then have been used as weapons. Although conferences were held to abolish chemical weapons, people still used them to fight each other. The trend of producing and developing chemical weapons went up over time. Continuing in the Cold War even new generations of agents were developed to harm others. Nowadays these chemical weapons are still present but difficult to trace. The German federal government does not make a major effort to investigate since the investigation is cost- and time-intensive. Despite the fact that these chemical weapons are maintained in our environment and society there are no specific systems or methods to detect them.

Therefore we had the vision to solve this problem by creating a detection system to identify contaminated sites which store chemical weapons. Our project P.L.A.N.T is a plantbased detection system that can detect contamination of specific chemical weapons in the soil. The detection can be seen visually through a change of its leaves colour. In our project we worked with chemicals that are related to the chemical weapons but do not contain any risks. These chemicals have been tested in Nicotiana benthamiana in a hydroculture which showed that the chosen chemicals are working. Furthermore we created a receptor that binds the weapon related chemical and we even optimized the signaling cascade. Besides that we used a RUBY reporter to show the visible change in the plant as a sign of detection.

During our project we worked with a lot of experts and stakeholders to develop this system. Another vision we had while working with these people was to create a community that new iGEM-Teams can take part of if they want to work with phototrophic organisms. Moreover we wanted to introduce synthetic biology to the general public hence we tried to impart the knowledge of ours to others as easily as possible. Because of that we developed “Ask Nici” as our Education Program to explain Biology to others easily.

Nici

Wiki Tour

Motivation


Introduction

In Germany, we have a problem that has been left behind by past generations. Spread all over the country, there are known as well as unknown storage sites of chemical weapon remains from both world wars. The reason for this is the insufficiently documented and uncontrolled disposal of Chemical Weapons [1]. In Germany alone, contaminated sites are suspected in over 200 locations [2][3] (map [1]).

However, tracing these sites is difficult, because the exact location and the dangers are not always known. Moreover, the German federal government makes no major effort to investigate these sites and to remove those chemicals. One of the key factors here are the costs: identification of locations and decontamination requires expensive equipment, time and effort [2][3].

Background

Since the middle of the 19th century, research in the field of chemistry has become increasingly important both for industry and for everyday life. However, the results of this research were not only used for the progress of these fields. The discoveries were quickly taken up by the weapons industry to develop even more cruel weapons for war [1].

The first time chemical weapons such as chlorine and phosgene were used was in World War I. Many countries started to research chemical weapons, which resulted in the discovery of new, toxic agents. One of those chemicals is lewisite, which was mass-produced in the USA. Another one is mustard, which caused more casualties than all other agents combined. Although between 1918 and 1933 several conferences were held where it was attempted to abolish chemical weapons, they were still used, for example against civilians in colonial possessions [4].

During the second world war, again a large amount of chemical warfare agents was produced. In Germany alone, 69.500 metric tons in ten factories [1]. Their development continued during the cold war, which resulted in the development of new generations of nerve agents. One of them is VX, which is three times deadlier than sarin. In the late 1980s, the USA started to destroy some of its stockpiles. However, the research continued on into the new millennium [4].

The destruction of chemical weapons in Germany as well as in other countries was insufficiently documented and controlled. This is the reason why significant amounts of the remaining chemical weapons were not disposed of properly. As a result, there are uninvestigated possible storage sites of chemical weapons from both world wars.

Another country that deals with similar problems is France. In the first world war, about one billion grenades were fired in France alone. Approximately fifteen million poison gas grenades did not explode. Each year, between twenty and twenty five tons of ammunition is found [5][6]. Further countries that have to deal with buried chemical weapons are the USA and Russia [7].

It is likely that due to huge costs of disposal, it will still take a long time until all known and suspected areas are decontaminated. During that time, there is a permanent risk: containers can become leaky so that chemicals that were made to cause harm, reach the environment. This risk is increased by storage sites that are newly discovered for example at construction sites which threaten to be ruptured before discovery [8]. These are great dangers that need to be dealt with [2][8][9].

However, at the moment there is no detection system that is suitable for screening and surveillance of large suspect areas on chemical weapon degradation products (human practices).

Our Vision

We made it our mission to solve this problem. We created a cost efficient and easy to use detection system with large area coverage to easily identify these contaminated sides. We believe that this will accelerate the process of decontamination.

The design and the proof of functionality of the detection system can be seen for the signaling cascade and the reporter RUBY. How we imagine our system to be used can be read at the implementation site. In addition, it is possible to adapt our plants to many other substances and pollution. How this can be performed is explained and proven at the computational design page.






















Our journey to P.L.A.N.T.

We introduce P.L.A.N.T., our plant-based detection system is designed to detect contamination of specific chemical weapons in the soil. If the plant comes in contact with their degradation products or precursors the plant signals it by changing its color to red.
As chemicals for our system, we chose methylphosphonic acid (MPA), diisopropyl methylphosphonate (DIMP), diethyl methylphosphonate (DEMP), thiodiglycol (TDG) and benzenetricarboxylic acid (BTCA). These chemicals are related to chemical weapons but safe to work with. They are stable in nature and have the potential to severely contaminate the environment and groundwater. For our plant-based detection system of these chemicals, it is important to test if they are absorbed by our plants and therefore are detectable.

Therefore, we cultivated Nicotiana benthamiana in a hydroculture and proved that the plants take up the chemicals DIMP, DEMP, MPA and TDG and transport these chemicals into the leaves. Additionally, we proved the uptake of BTCA, which is safe to work with and has a similar structure to TNT, for which a corresponding receptor for the detection is available. The detection system is based on a receptor which binds the chemical, the activation of a signaling cascade upon ligand binding and resulting in the expression of a reporter.

Crucial for the functionality of our chemical weapon degradation products detection system is a receptor which is able to bind the desired chemical. By applying computational protein design on a ribose binding protein, we created a functional and specific receptor to bind a weapon related chemical. By using a modular system, the receptor can be changed easily to create a new plant for detection of further chemicals. One way to do this is the computational design we successfully performed and therefore can present a modeling pipeline that we used to successfully engineer proteins (Parts: BBa_K3900001, BBa_K3900002, BBa_K3900003, BBa_K3900004, BBa_K3900005, BBa_K3900006).

Before implementing our designed receptor in the signaling cascade, we first tested it in Escherichia coli and proved its functionality (Parts: BBa_K206000, BBa_K216004, BBa_B0014, BBa_K216003, BBa_K808000, BBa_R0082, BBa_E0040, BBa_P1003, BBa_K731722). Afterwards, we optimized the signaling cascade by using our designed receptor, which was able to bind its ligand (BTCA, BBa_K3900002).

After detection of a chemical weapon degradation product and the activation of the signaling cascade, a visible output is created. Therefore, we introduce the RUBY reporter, which codes for betalain synthesis genes, and creates a visible red coloring of the plant. In addition, we adapted the ANTHOS reporter, which is based on the synthesis of anthocyanins (Parts: BBa_K3900028, BBa_K3900046, BBa_K3900047) .

To perform initial tests with our signaling cascade and our newly engineered receptors, a fast growing and easy manipulative model organism was required. Therefore, we worked with Escherichia coli for first testing of a bacteria adapted signaling cascade and the engineered receptors. When we proved its functionality, we tested the plant signaling cascade by transient expression in Nicotiana benthamiana.

In order to develop and improve P.L.A.N.T., our plant based detection system for chemical degradation products, we were in constant contact with different experts and stakeholders throughout the year. With their help, we were able to adjust the aim of our application to the detection of degradation products for identification of contaminated sites and to further fine-tune our experiment project realization.

However, we did not only talk to different experts, we also talked with other teams working with plants. Our vision was to create a community that new iGEM-Teams that want to work with phototrophic organisms can join to connect, get help, learn and teach. Our goal is a lasting community that not only stands behind new teams but also generates, over the years, great collective knowledge in the form of our Phototrophs-Handbook. This way we will be able to help future iGEMers and scientists working with phototrophic organisms from all around the world. The fact that synthetic biology is unknown to a large part of the population and lacks decades of experience makes it difficult to deal with in terms of regulation, social acceptance and ethics. Our goal is to introduce the general public to synthetic biology. To accomplish that, we talk about the basic concepts of biology to give everyone the opportunity to understand synthetic biology and to show how it can be used to help humanity in a sustainable and efficient way. Therefore, we have set up a project called ‘Ask Nici’, our education podcast.

References



1. Preuss, J. The reconstruction of production and storage sites for chemical warfare agents and weapons from both world wars in the context of assessing former munitions sites. One Hundred Years of Chemical Warfare: Research, Deployment, Consequences. Springer, Cham, 289-333 (2017).

2. https://www.tagesschau.de/inland/chemiewaffen-altlasten-deutschland-101.html (access 02.10.21)

3. https://www.presseportal.de/pm/118095/4679184 (access 01.10.21)

4. Evison, Demetrius, David Hinsley, and Paul Rice. "Chemical weapons." Bmj 324.7333: 332-335. (2002)

5. https://info.arte.tv/de/welche-gefahren-gibt-es-weltweit (access 01.10.21)

6. https://robindesbois.org/en/comment-la-france-gere-t-elle-les-munitions-chimiques-anciennes/ (access 01.10.21)

7. https://journals.sagepub.com/doi/10.2968/057005014 (access 03.10.21)

8. https://www.chemistryworld.com/features/the-great-war-clean-up/3009456.article (access 03.10.21)

9. https://www.dw.com/en/a-deadly-legacy-chemical-weapons-in-germany/av-56502637 (access 02.10.21)