Summary

Background

Food security is an urgent global issue. It is predicted that the food demand will continue to rise as the population increases, leading to serious problems such as resource depletion and agricultural land reduction.

The effects are already being felt today, with 9.8% of people facing hunger in 2021, according to the FAO [1].

One reason for this problem is agricultural damage from wild animals. Environmental changes and disruption to their ecosystems push these animals out of their natural habitats, causing their effect on agriculture to increase.

Fig.1 Crop damages by deer

Agricultural damage from wild animals can be commonly seen in Japan, and almost half of those damages are caused by deer. (a) Damage to broccoli from deer [2].
(b) A deer eating watermelons in Hadano City, Kanagawa Prefecture.[3].

The USDA reported in 2022 that the total damages to livestock and crops caused by wild animals in the US was over $380 million, threatening the safety and health of humans [4].

In Japan, deer are the main cause of agricultural damage by wild animals. In 2021, deer-related agricultural damages made up about half of the total damages from wild animals, with the damages amounting to $40 million being the largest out of all wild animals [5] [6].

Fig.2 Amount of damage to crops

Agricultural damages from wild animals in Japan total about $100 million every year [7], with damages from deer comprising about 40%.

Typically, physical methods such as electric fences are used to prevent deer damage, as in Fig.3. However, installing and maintaining fences incurs high costs, and there are also areas where fences cannot be installed. In addition, because deer have a high jumping ability, they may jump over fences. Some methods use sound, ultrasonic waves, and light, but these also have their limitations, and few of them have been proven effective and are readily available to farmers.

Fig.3 Example of electric fence installation

A field in Iwakura, Kyoto protected by an electric fence.

Our Project

As a solution to the deep-rooted problem of crop damage by wild deer, we propose the use of E. coli capable of long-term production and dispersion of deer repellent substances in the field.

Our project involves the development of a method for extended E. coli cultivation and designing a non-electric, non-gas-powered incubator.

We are confident that in the future, crop damage caused by deer could be controlled by

1. Production of repellents against deer

We have selected 2-phenylethylamine (2-PEA), a compound identified in bobcat urine and known to induce fear responses in mice. Lion urine has been reported to contain comparatively more 2-PEA than other carnivores [8] and is the only member of the Felidae family whose urine has a consistent repellent effect against deer [9]. There have also been attempts to use lion urine to keep deer away from train tracks [10]. From these studies, we can reasonably hypothesize that 2-PEA has a strong repellent effect against herbivores like deer.

Using scents to drive away animals is an effective strategy rooted in nature. For example, rodents have shown avoidant behavior against 2,4,5-trimethylthiazoline (TMT), a component in fox excrement [11]. This reaction is encoded in neuronal circuitry of the olfactory system, including olfactory epithelium and amygdala. These studies show the variety of molecules that can induce fear responses and the importance of effective production and dispersion of these repellents.

2-PEA has previously been shown to be produced in E. coli [12] [13], providing us with a usable system for our synthetic biology chassis. From data about the deterrent effects of lion urine on deer, we were able to calculate the minimum concentration of 2-PEA to deter deer, and the corresponding E. coli culture size to reach this amount (see Model Part 6 for details). Thus, the only task remaining is building a structure to make E. coli produce 2-PEA consistently over a long period. The next chapter will explain our process in designing the E. coli to achieve this, as well as our ground-breaking hardware that enables an eco-friendly way to culture these new E. coli (See Hardware for details).

2. Stabilization of the long-term cultivation

Existing E. coli bacterial cultures are typically optimized for short-term expression during the log growth phase, making them unsuitable for the desired long-term expression needed to achieve a long-term effect. Moreover, substantial nutrient resources in the culture medium are diverted to non-product molecules, notably the bacterial mass of E. coli, resulting in significant resource loss. By strategically designing the culture to limit its population density and recycle substances from dead E. coli, thereby restricting resource consumption from the medium, we plan to ensure prolonged production of target substances like repellents.

We have crafted three key strategies to attain this:

These approaches aim not only to enable the prolonged cultivation of E. coli and the sustained expression of target compounds but also to bolster the efficient use and utilization of nutrients by recycling lysed E. coli components, reducing waste. (See Results and Model for details).

(a) Quorum sensing and lysis

Our system to limit E. coli population density took inspiration from iGEM Ecuador 2021 [14]. Their project is based on RNAi to control viral infections in plants via a combination of quorum sensing (QS), a phenomenon wherein the expression of an individual changes according to the population density, and a bacteriophage-derived lysis gene. We took inspiration from their strategy of population control and applied it to our goal of long term protein expression.

By adjusting E. coli behavior according to the population density, we aimed to keep the number of E. coli cells below a certain critical value, therefore avoiding unneeded nutrient consumption and allowing for long-term expression (See Results and Model Part 1 for details).

Fig.4 The QS-Lysis circuit

Design of the gene circuit with both quorum sensing and lysis circuits, inspired by iGEM Ecuador 2021's design. The goal of this circuit is to keep the E. coli population below a certain value, thus allowing for continuous, long-term expression.

(b) Nutrients Recycle System

We found that the lysed E. coli in this system contained nutrients that could be recycled. The components of E. coli have been studied as shown in the following table [15].

Table 1. E. coli composition by weight

Values are relative to the dry weight of an E. coli cell. Our team focused on recycling the protein and nucleic acids, which are not naturally recycled, but compose 80% of the cell's dry weight.

In a lysis system as in 2-a, the contents of E. coli get released into the culture [16][17]. The released matter is composed mostly of proteins and nucleic acids, which are both materials that can be reused as nutrients [18][19][20]. By reusing the proteins and nucleic acids of lysed cells as nutrients, the nutrients in the broth can be maximized, making long-term culturing easier to achieve.

However, proteins and nucleic acids cannot be used as nutrients as is [21]. Thus, to support our main goal of long-term culturing, we aimed to incorporate protease and DNA/RNA endonuclease [22][23] into the system to maximize the nutrients from lysed cells' remains (See Results and Model Part 4 for details).

Fig. 5 The nutrient recycling system

Benzonase (DNA/RNA nuclease) degrades DNA in the cell and subtilisin (protease from Serratia marcescens) breaks down proteins in the medium by being secreted out of the cell.

(c) Differentiation System

Due to reduction in fitness, cell death systems, including the lysis system mentioned above, are subject to high selective pressure, and thus will be easily mutated [24][25]. For example, if a mutant that deactivates the QS circuit connected to the lysis circuit above appears (a loss-of-function mutant), its naturally high survival advantage will cause this mutant to quickly become dominant, making the culture lose the desired “disadvantageous” cells.

To address this, we introduced a system that will allow part of the population to “differentiate” and exhibit the lysis function described by Williams & Murray (2022) [26]. Because the replication of the R6K plasmid encoding essential genes is halted in differentiated cells, the number of R6K plasmids per cell will decrease as these differentiated cells divide. Because a decrease in R6K plasmids means the loss of essential genes, there is an intrinsic limit to the number of divisions a differentiated cell can do. Even if a mutation disables the lysis gene and a population of non-lysogenic mutants appears, these mutants will not increase above a certain value. With this system, the selection pressure against burdensome gene circuits like the lysis circuit can be alleviated, and the length of expression can be increased (See Results and Model Part 2 for details).

Fig.6 The cell differentiation system

(a) When integrase is not expressed, LuxR is split into two, and quorum sensing functions are turned off. The pir protein facilitates R6K origin plasmid replication, and essential genes (EG) are expressed normally.
(b) When integrase is expressed, the cell is “differentiated”, LuxR is reformed by recombination, and quorum sensing functions are turned on. At the same time, because there is no pir protein, R6K origin plasmid replication is turned off, and an intrinsic limit to the number of replications is placed.

Hardware

We have designed a device that can culture E. coli that produces the odor-repellent 2-PEA with minimized energy consumption. This device is based on a traditional Japanese device made of bamboo that was traditionally used to drive away deer, the shishiodoshi (or shikaodoshi, literally “deer scarer”), and an old toy called the drinking bird. Using the principles [27] from the latter, this device can autonomously continue its own motion unattended for about a month (See Hardware and Model Part 6 for details).

Fig.7 Hardware implementation plan

Compared to the usually used electric fences, this device's initial cost and maintenance cost are cheaper, making it a viable solution to deer-related agricultural damages (See Human Practices for this device's societal impact)

Table 2 Comparison of our device and electric fences as deer deterrents

Legend: yellow - excellent, red - good, blue - undesirable. Our device was particularly designed to minimize costs and maximize ease of use.

Aside from culturing E. coli that produce repellent molecules in the field, this device also has advantages in culturing cells in the laboratory, mainly from its low energy consumption.

We have succeeded in using this device to culture E. coli powered by just the heat of vaporization of water, instead of more traditional batteries or circuit boards. We opted to avoid these materials because of the environmental cost involved in their production, as well as their chances of causing additional damage to the environment when left in the wild. Thus, this new device does not only make more ethical culturing in the lab possible but also expands the possibilities for culturing bacteria in the fields.

Due to safety concerns, we only conducted experiments in the laboratory this year, but once the safety concerns have been resolved and we are assured that our experiments will have no negative effect on the environment, we plan to test if our device can continuously culture bacteria in the fields. (See Safety for details.)

Find out more

Results

                 

Engineering

                   

Hardware

                  

Human Practices

                  

References

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