Overview

Our project aims to use E. coli to repel deer, which eat and destroy crops and forestry resources, from fields and parts of forests. Specifically, we will use genetically modified E. coli to produce a repellent to deer (2-phenylethylamine; 2-PEA) and release the molecules in fields and parts of forests. The first phase of the project will involve producing 2-PEA in the laboratory, but the ultimate goal is to install hardware in forests and by fields to produce the repellent molecule by E. coli on site.

To realize this project, we planned field and lab experiments and implemented thorough safety controls for both aspects.

As a result, we had to scale back our experimental design twice due to several hazards. As a result of these changes, our experimental design is now extremely safe. Even when performing lab experiments, we took the utmost care to ensure safety by attending a GMO course and being directly supervised by our experienced PI.

Field Experiments

Overview

We first designed a field demonstration experiment using actual genetically modified E. coli. The experiment consisted of three parts:

However, we decided to exclude the first step from this year's plan at the beginning. This is because experiments to cultivate genetically modified E. coli outside of the lab violate many safety guidelines, and we judged careful safety studies over a long period of time were essential before conducting the experiments.

We also decided not to work on the second step this year. We planned to purchase chemically synthesized 2-PEA, and after consulting with Dr. Ishihara from Ashiu Forest Research Station at Kyoto University, we also planned an experiment to volatilize it in the forest. Since we would not use genetically modified organisms in this experiment and would observe wild animals, we were not included in the Regulations on Animal Experimentation at Kyoto University [1]. However, as we were preparing the safety sheet for iGEM, we decided to postpone this experiment as well, because we found that the molecule has not been proven to be completely safe.

Based on the above, we have decided to focus our experiments this year on "3. Developing a system to efficiently synthesize 2-PEA in the laboratory".

However, these decisions do not mean that our project to produce 2-PEA with E. coli grown in the field is impossible. We expect that with the confirmation of the safety of 2-PEA, the selection of other, safer repellent molecules, safe hardware that does not leak the bacteria, and various improvements, our device will reach the people who need it in the field in the future.

1. Field demonstration using GMOs

Our Plan

We initially planned an experiment in which we would cultivate the genetically modified E. coli in the hardware we had created and actually place the device in Ashiu Forest Research Station to verify its effectiveness.

Hazard Considerations

We discussed the dangers involved when conducting field demonstrations. As a result, we decided that containment of the organisms was necessary and that there was a possible risk that our genetically modified E. coli could be spilled out in the unlikely event. We considered using hardware-based biocontainment to address this risk.

Biocontainment in Hardware

Our hardware allows E. coli to be cultured by concussion inside a capsule as the one pictured, and releases 2-PEA, a deer repellent, through the holes on the top of the capsule. However, with the holes on the capsule, there is a possibility that genetically modified organisms could leak out if animals in the natural environment kick it or if this hardware were to topple over due to impact from natural phenomena such as rain, wind or earthquake.

Fig.1 The concept of our hardware device

The device is placed in the forest and the E. coli inside is cultured by concussion. The device releases 2-PEA, a deer repellent, through the holes on the device.

Final Decision

In addition to the risks associated with this hardware, there are the risks of mutation. Our project is focused on the long-term cultivation of the bacteria. In this process, as E. coli continues to proliferate, mutations are inevitable. As a result, mutant strains that are resistant to the kill switch must emerge [2]. We have discussed these issues with our PI, taking into account the Cartagena and Japanese laws, as well as the negative impact on biodiversity and ecosystems. As a result, we concluded that the risk of the E. coli spill far outweighs the benefits expected from the project at this stage, and we decided to reduce the scale and devise a new plan.

2. Field demonstration using 2-PEA

Our plan

Based on the above reduction, we decided to purchase 2-PEA, the substance we wanted our E. coli to produce, from a company, create hardware to release the substance, actually install the hardware in the research forest, and quantify the degree to which deer would find the device repellent.

Hazard Considerations

In the course of our planning, we considered several dangers. These include (a) ethical and legal issues, (b) the negative impact of using chemicals in the field on biodiversity and ecosystems: environmental impact, and (c) the effects on the human body.

(a) Ethical and Legal Issues

We discussed these issues with Dr. Ishihara. We concluded that the ethical issues could be resolved by limiting the location of our hardware to a portion of the forest, rather than the entire area, so that the deer would have a place to escape, and by using unattended cameras to observe deer behavior so as not to interfere with their autonomy. We also referred to the Japanese law on the protection of birds and animals and confirmed that our experiment did not conflict with its regulations[3].

(b) Environmental Impact

We investigated the use of 2-PEA in the natural environment to see if it would have any significant impact on biodiversity and ecosystems. We discussed the dangers of the molecule with Dr. Ishihara. As a result, we confirmed based on previous research that 2-PEA is actually present in the environment as it is abundant in the excrement of carnivorous animals[4]. This led us to conclude that the damage from exposure to the environment would probably not be significant. We also rechecked the ThermoFisher SCIENTIFIC safety data sheet on 2-PEA to see if it was indeed safe[5], and although 2-PEA was confirmed to be persistent, the data sheet did not contain any information on environmental effects, which we judged to be insufficient information.

(c) Effects on the Human Body

We also checked the safety data sheet for its effects on the human body. As a result, model experiments were conducted on mice, which showed that the LD50 for oral inhalation was 400 mg/kg. We judged it to be somewhat dangerous, although 2-PEA did not fall into either of these categories, since the definition of a toxic substance is an LD50 of 50 mg/kg or less and the definition of a deleterious substance is between 50 mg/kg and 300 mg/kg. Also, no data was available in this safety data sheet regarding dermal inhalation.

Final Decision

Based on the above, we concluded there may have been some danger, and there was not enough safety information regarding the environmental and human health effects of 2-PEA. With the level of completeness of our project at this stage and the inadequate data obtained from our in-lab experiments, the benefits to be gained from a demonstration experiment would not outweigh the dangers of the environmental and human health effects of 2-PEA. Finally, we decided to limit our experiments to in-lab testing.

Safety in the Laboratory

Biocontainment

The organisms we used were E. coli DH5α, BL21, and BW25113 (provided by the National Institute of Genetics, Japan), all of which are classified as Risk Group 1. Thus they are not likely to cause human disease and are also included in the white list of organisms. We also conducted all experiments related to this project in our laboratory. Waste liquids containing genetically modified organisms were clearly distinguished so that they could never be spilled by mistake, and when disposed of, they were autoclaved based on the advice of our PI or properly treated with chlorine bleach if necessary. In addition, all tips, tubes, and other paper and plastic waste were properly separated and disposed of as experimental waste. Furthermore, all plates that touched genetically modified organisms were autoclaved before disposal to ensure that live genetically modified organisms were never released. Since we were conducting our experiments in two labs, we had the opportunity to transport colony-grown plates from lab to lab during the course of our experiments. In accordance with iGEM's Safety Policies[6], we taped the plates tightly and carefully transported them ourselves.

Fig.3 Our Treatment of Genetically Modified Organisms

E. coli strains were transported securely between labs, with plates tightly taped to prevent any live genetically modified organisms from being released during transit.

For safe experimentation

We were directly instructed by our PI on how to safely conduct experiments prior to performing them, and we reviewed equipment and practices that could result in death or injury. We received direct instruction on how to handle dangerous experimental maneuvers, e.g., liquid nitrogen, gas burners, autoclaves, and centrifuges. We also considered the dangers of dangerous chemicals. We used Ethidium Bromide (EtBr), a suspected carcinogen, for DNA electrophoresis and received direct instruction from our PI on how to handle this substance. We did not use any other strong acidic or basic substances, corrosive substances, substances that cause damage to the central nervous system, heavy metals, mutagens, explosive substances, and similar potentially hazardous chemicals in this experiment.

Experimental Training

Before conducting our genetic modification experiments, we attended a DNA modification course at Kyoto University. Here we learned about the laws and regulations governing the use of genetically modified organisms and how they should be handled and processed. We also learned about the past cases of improper use of genetically modified organisms and emergency situations, as well as countermeasures against them.In addition, our PI had extensive experience in experiments with genetically modified organisms, including the E. coli we would use, and we received direct instruction on the safe handling of genetically modified E. coli by him in the actual lab.

References

[1] Kyoto University(2022), Regulations on Animal Experimentation at Kyoto University. https://static.igem.wiki/teams/4655/wiki/regulations-on-animal-experimentation-at-kyoto-university.pdf

[2] You, L., Cox, R. S., Weiss, R., & Arnold, F. H. (2004) Programmed population control by cell–cell communication and regulated killing. Nature, 428 (6985), 868-871. https://doi.org/10.1038/nature02491

[3] (Japanese) Ministry of the Environment (2002), Wildlife Protection, Control, and Hunting Management Act https://www.japaneselawtranslation.go.jp/ja/laws/view/3736

[4] Ferrero, D. M., Lemon, J. K., Fluegge, D., Pashkovski, S. L., Korzan, W. J., Datta, S. R.,..., & Liberles, S. D. (2011). Detection and avoidance of a carnivore odor by prey. Proceedings of the National Academy of Sciences of the United States of America, 108(27), 11235-11240. https://doi.org/10.1073/pnas.1103317108

[5] Thermo Fisher Scientific (2021), SAFETY DATA SHEET, 2-Phenylethylamine. https://static.igem.wiki/teams/4655/wiki/2-phenylethylamine-hydro-25gr.pdf

[6] iGEM (2023), Release Beyond Containment, Safety Policies, Responsibility, iGEM. https://responsibility.igem.org/safety-policies/release-beyond-containment