Introduction

Safety and security have always been at the center of discussion in the field of synthetic biology. Safety focuses on protecting the human body and the environment from genetically modified organisms (GMOs), and security focuses on protecting genetic information and features of GMOs from nefarious interests. For example, Strack et al. (2008) introduced an example of a biological security system using the order in which substances are administered [1]. The system introduced in this paper is based on the reaction of biomolecules, so it is difficult to combine it with non-molecular signals such as light. In addition, past iGEM projects worked on safety and security measures, such as HKUST 2017 which created a biosafety system to put a time limit on gene expression with recombinase [2], and NEFU_China 2018 which created a security system to encode English text into DNA sequences and lock them [3]. We were inspired by their use of recombinase, the insertion of introns, and the creation of dummy sequences for creating a security system. Yet, while these made very important steps, they have not been able to achieve safety and security at the same time.

Optopass can solve both safety and security challenges simultaneously. By using the order of light as a cryptogram, we can ensure safety with the light inducible kill switch and security with the high complexity of the light sequence.

Biosafety

Biosafety

The Optopass system is useful in expanding the use of synthetic biology. One of the concerns about synthetic biology is the release of GMOs outside of controlled environments such as laboratories and production lines. The potential problems a spill of GMOs can cause are wide-ranging. For example, a gene flow from the GMOs to organisms in the environment may occur. There is also the danger of competitive exclusion of native species and the production of toxic substances from GMOs. The risk they pose of damaging biodiversity and destroying ecosystems is not small. Biosafety is essential in the process of ensuring that synthetic biology can spread widely and safely to the public. This concern is shared worldwide, and the Cartagena Protocol on biodiversity, which aims to make biotechnology safe and useful to society, has been adopted by countries all over the world to build consensus. However, some countries have not ratified it, such as the United States and Australia.

Although the global guideline for synthetic biology is not yet completed, some of the techniques are already being used by companies. We have learned through human practices that Japanese companies are very concerned about compliance with the Cartagena Protocol. When scaling up from the lab to the production line, though there are many things to keep in mind, the biggest challenge is the inactivation process, which must kill GMOs without fail. Currently, heat treatment is mainly used, but there are several problems with this method. First, heat is likely to denature substances. In industrial production lines, synthetic biology techniques are often used to make microorganisms produce organic compounds which can be thermally denatured, so heat treatment is not an industrially performant method. It is also dangerous to humans in that humans can be hurt by the heat. Furthermore, we found out that equipment maintenance costs are high due to heat damage. For these reasons, the heat treatment widely used today is not ideal.

On the other hand, the Optopass system from UTokyo solves this problem. Optopass is a system that allows microorganisms to produce the target substance only when exposed to light in the correct order and kills the microorganisms when exposed to the wrong light or sunlight, which means that GMOs are inactivated by their exposure to the incorrect light or sunlight. Therefore, sterilization can be accomplished just by passing them through a fixed length of pipe under light irradiations, eliminating the need for high-temperature treatment and leading to simplified maintenance. What is more, GMOs could be killed with easy and accurate stimulus control. Optopass is a cheaper and simpler inactivation system that eliminates hazards in industrial production lines and provides a powerful boost to biosafety.

Furthermore, synthetic biology can give microorganisms great power. We believe that the potential of synthetic biology will be expanded when we realize "synthetic biology of the people, by the people, for the people." The most important thing to be aware of when using synthetic biology in citizen science as a solution for problems would be the leakage into the environment during its use. This is of course also true when handled by companies and researchers, but the risk of human error is particularly high for citizens with relatively low proficiency in biological experiments, so countermeasures are important. This is one of the reasons why synthetic biology is not often addressed in citizen science.

Optopass can collectively solve these challenges to make synthetic biology safely available to citizens. In addition to the aforementioned convenience of using light as the final treatment, Optopass will kill microorganisms under the sunlight by its kill switch. This is because the kill switch is placed under light inducible promoter control in Optopass. When GMOs are exposed to sunlight, composed of a wide range of light wavelengths, they will interpret that they are exposed to the wrong light and commit suicide. Therefore, even if GMOs unintentionally leak out, they will not be able to survive in the environment once they are exposed to sunlight. The risk of GMOs’ genes being taken up and working in other organisms is also considered low because the genes are fragmented when the kill switch is triggered. Since the effects of genome editing on ecosystems are unknown, light inducible kill switch is an essential function.

Experimental Safety

The key mechanism of the Optopass system, which uses the order of light as a stimulus to control organisms, ensures not only biosafety, but also safety during experiments. Light is a safer and easier-to-handle stimulus compared to heat or chemicals. In citizen science, ultra-high temperatures and chemicals are difficult to use from a standpoint of equipment, processing, and permitting requirements. Even labs that can treat chemicals must be careful about the post-treatment of chemicals, and learning disposal measures can be time-consuming and difficult. Optopass improves safety for both citizen science and laboratory experiments because the system uses light, a tractable stimulus.

Biosecurity

Context & Design

Pharmaceuticals, biofuels, and flavorings are among the many products manufactured using synthetic biology. DIY biotechnology activities indicate that synthetic biology is gaining popularity among the general public. While these facts foreshadow the development of biotechnology, the increasing sophistication of research and industrial use of synthetic biology introduces new risks, such as biotechnology theft and the simplified development of biological weapons. Unintentional technology leakage is becoming a reality, with disastrous consequences for the military and security, along with threats to everyday life.

In this situation, governance and transparency of the technological development process are critical for science and technology to develop with public understanding. To counter theft by malicious third parties, including its diversion to biological weapons, security measures beyond legal constraints must be considered. Optopass was conceived in this context.

In addition to the suicide mechanism described above, our design combines light inducible promoters with recombinase systems to create an 'optical passcode', a mechanism for controlling gene expression using light inputs. The desired substance is produced when the correct colors of light are shone on the yeast in the correct order; otherwise, a kill switch is triggered, and the yeast commits suicide. This security system, using order, can lead to stronger security compared to using a combination of lights. For example, when three colors of light are used as stimuli, only a maximum of seven patterns can be created in combination, but when the cipher length is set to 10, 1,536 candidate patterns can be created in order. Combining light and order may provide an easy-to-use and robust security. Furthermore, Optopass, which locks the organism itself, has the potential to protect genetic information more directly than existing methods that lock the organism's storage, which means that Optopass can be a more assured security system than the current ones.

Bioterrorism

Bioterrorism and the proliferation of biological weapons are the most well-known threats in the field of biosecurity, as demonstrated by the "anthrax attacks" in the United States in 2001, in which anthrax taken from a laboratory was used to commit terrorist acts and caused deaths. To prevent such heinous terrorist acts, research, technology, and biological agents with dual-use characteristics – that is, things that can be used for military or terrorism purposes even if they are intended for civilian use – must be identified and properly managed to prevent them from being inappropriately leaked. The Royal Netherlands Academy of Arts and Sciences has proposed a code of conduct for biotechnology and research to prevent diversion to bioterrorism through appropriate controls [4]. Each researcher can fulfill their obligations under the Biological and Toxin Weapons Convention (BTWC) and prevent technology leaks that could lead to terrorism by adhering to this code of conduct. The code of conduct is divided into six sections: raising awareness, research and publication policy, accountability and oversight, internal and external communication, accessibility, and shipment and transport. Optopass is an effective tool for bolstering measures in three of the six sections of the code of conduct.

Raising Awareness

According to Prof. Shinomiya at the National Defense Medical College (See Integrated Human Practices page), all laboratories in Japan have a high level of biosafety awareness due to the Cartagena Act, but biosecurity awareness is still in its infancy. Optopass is a solution for not only biosafety, but also biosecurity; therefore, by creating a product that combines biosafety and biosecurity utilizing Optopass and popularizing it through the high level of biosafety awareness, biosecurity awareness can be raised.

Accessibility

Optopass requires the optical cipher in order to utilize the actual biological agent. Therefore, access to the biological agent necessitates access to both physical material and the cipher. Previously, access to the physical material directly meant access to the biological agent's use. However, if Optopass is installed and the biological agents and optical code are managed separately, access to the biological agents can be restricted to those with the physical access and knowledge of the optical code. This reduces the possibility of malicious individuals gaining unintentional access to biological agents.

Shipment and Transport

Optopass inputs an optical cipher in biological agents and is a photo-inductive kill switch. This means that even if the biological agent is stolen during transportation, the third party cannot use it, and even if it leaks into the environment while it is transported, it will be killed by light, preventing any impact. Furthermore, by separating physical transport from code transmission, only those with access to both the agent and the code, typically the researchers at the destination who have the right to access the biological agent, will be the only ones who is able to hand over the biological agent to a malicious third party. This limits the number of people who can release the bioagent, reducing the risk of spillage from the destination of the bioagent.

Industrial Espionage

The gene expressions that we want to protect from threats are not just those that are directly dangerous, such as the ones that have the potential to be diverted to bioweapons and bioterrorism. Organisms that produce materials as the base of advanced industry are constantly threatened by industrial espionage.

If companies want to protect the economic benefits that derive from material-producing organisms (here we call them intellectual property) from threats, they have two options: they can either file the organisms as patents or keep them confidential as trade secrets. If a company files a patent, they gain a technological monopoly in exchange for making the organism's genetic information available to the public, and they can sue for monetary damages if anyone uses it. Trade secrets, on the other hand, do not give rise to a monopoly on the technology, so others who have developed the same technology can use it without any restrictions.

We conducted research and found that, when intellectual property is protected as trade secrets, the biggest issue is industrial espionage, which is an attempt to gain a competitive advantage by stealing trade secrets. Even though there are criminal penalties for theft, they are not always imposed because the competitor can be a company in another country or the stealing technique is so sophisticated that it cannot be detected, and there are many cases of economic disadvantage due to trade secret leakage. Measures against industrial espionage are urgently needed in order for science to develop under the proper economic activities of companies.

So, what steps should be taken? Essentially, the framework is the same as that taken against bioterrorism measures. First, by developing a security system that works in tandem with our biosafety assurance system, we are able to raise awareness of industrial espionage measures among new companies entering the field of industrial production of substances through synthetic biology, because they have to comply with biosafety. Second, by separating the physical and cryptographic control of microorganisms, it is possible to specifically limit who can take and use the microorganisms compared to protecting just the physical organism. Third, even if the biological agent is stolen during transport, the thief will have difficulty using it because it is equipped with a light inducible kill switch and it will kill itself once it is exposed to sunlight or the light of a wrong wavelength.

According to Kaneka (See Kaneka section in Integrated Human Practices page), industrial cultivation in companies is carried out first by cultivating small quantities of strains stocked in laboratories to about 1 litre in the laboratory and then by transporting them to industrial culture tanks of 1 tonne or more. The industrial culture tanks are subject to strict leakage controls in accordance with the Cartagena Protocol, lowering the risk of leakage in the process. Security should thus be focused on stock storage in the laboratory as well as the transport process from the laboratory to the culture tanks. As stated above, Optopass can make a significant contribution to both of these processes by its killswitch.

DNA Storage

The Optopass system protects more than just GMOs’ genetic information. We are investigating the possibility of combining optical passcode systems with DNA storage, which is a technology that attempts to convert binary data into nucleotide sequences for storage. [3] Securing the physical place where the DNA is stored is a commonly considered method of protecting DNA storage, but with Optopass you can directly protect the DNA sequence itself; Optopass could also contribute to the security of various types of data.

DNA storage has a longer record retention period and higher recording density than existing recording technologies like HDDs, but it also consumes time to read it because sequencing is required, and is thus likely to be used in large data centers to store large amounts of data for long periods of time. Shiori Inoue, from the Bank of Japan, describes the information handling process in relation to security in large data centers as follows.

[Process of writing]

1. Apply the conversion rules to the binary data brought in by the customer on the data conversion terminal in the office and convert it into a nucleotide sequence.
2. Save the obtained nucleotide sequence on a USB memory stick and bring it to the laboratory, connect it to a DNA synthesis machine and make a copy.
3. Use the DNA synthesis machine to synthesize the DNA and primers of the customer's data.
4. Keep the Synthesised DNA and primer sets in a warehouse.

[Process of reading out]

5. Remove the DNA and primer set from storage and amplify it with a DNA amplifier.
6. Use a sequencer to perform sequence analysis.
7. Save the sequence analysis results on a USB memory stick, bring it to the office, connect it to the data conversion terminal for data conversion, and copy the results.
8. Use the data conversion terminal to convert the sequence analysis results into binary data.

In light of this, there are two methods for stealing data: 1) estimating the sequence of synthesized DNA based on noise emitted by an operating DNA synthesis machine [5], or 2) taking the DNA stored in the warehouse.

The Optopass's gene circuit allows recombination to occur in different ways depending on the order in which the light is shone. Therefore, for 1), we can make a gene circuit by Optopass, where even if all of the nucleotide sequences are known, the target sequence cannot be obtained if the order of light exposure is incorrect. For 2), the kill switch in Optopass breaks the DNA apart when exposed to white light, so when the DNA is extracted, it falls apart and cannot be read. Furthermore, as previously stated, if the order in which the light is shone is unknown, even if stolen, the target sequence cannot be obtained.

Dummy System

As a follow-up to Optopass, we created the Dummy System. A target strain (carrying the gene of interest) and a dummy strain (without the gene of interest, but with an optical passcode system) are prepared in the Dummy System. The optical stimulus controls the equilibrium or imbalance in the competitive relationship between the two strains, so that if the input is correct, only colonies of the target strain are obtained, whereas if the input is incorrect, only a colony of the dummy strain is obtained. When the two strains are coexisting, the sequence of the target strain is protected by the camouflaging effect of the dummy. The goal of protecting the target strain's genetic information is similar to that of the standard Optopass, but where the standard Optopass can only protect it by controlling the expression of a single gene, the Dummy System can protect the entire genetic information of the target strain through the camouflaging effect of the dummy. It will also be able to protect strains of those in which Optopass is difficult to introduce genetically. This may meet a company's need to safeguard its valuable strains. Furthermore, the Dummy System employs competitive exclusion, which can kill target microorganisms more reliably than the kill switch, resulting in increased security.

Laboratory Safety

Working safely in the laboratory is essential thing in ensuring biosafety. iGEM UTokyo has always conducted our experiments in accordance with the safety policy of the iGEM committee and the University of Tokyo.

Before starting the lab work, team members got training on how to treat GMOs and their risks. Year 2 students also took a course "Introductory Experiments (Biological Science)" this spring semester, where they learned the basics of laboratory experiments. Once the experiment started, we always submitted our experiment plans to the lab every week to ensure that the experiment was conducted safely. We also conducted the experiment in the presence of a lab researcher to prevent the risk of leakage of GMOs. The working members of the wet lab varied from day to day, but we ensured that at least one of the members who had more than half a year of wet lab experience supervised the work for biosafety reasons.

In our project, we only worked with organisms from risk group 1: Escherichia coli (JM109) and Saccharomyces cerevisiae. Both of them are non-pathogenic strains.

Yet, S. cerevisiae had a higher risk of contamination than E. coli because it also spreads by spores. For this reason, we used disposable incubators for S. cerevisiae, and when culturing on agar medium, we wrapped the plates in plastic wrap to prevent spores from spreading. Materials containing GMOs were always autoclaved. Effluent was managed in separate containers for S. cerevisiae and E. coli, and appropriate treatment was given to each type of bacteria. Precautions for the risk of chemicals such as ethidium bromide were also taken.