Overview

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Safety is one of the fundamental principles of scientific research. The development of safety regulations is aimed at protecting all individuals, including scientists.

In this competition, iGEM Tianjin aimed to promote the advancement of biosafety-related technologies and developed a kill switch system based on epigenetic modification called the Wind-up Cell system.

Our experimental designs strictly adhere to safety principles, and all members undergo rigorous experimental training before entering the laboratory. Additionally, we conducted biosafety awareness campaigns to enhance public awareness of biosafety.

Figure 1. Five parts related to security for the iGEM Tianjin’s project

Project and Biosafety

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With the continuous development of technology, synthetic biology is gradually entering our lives, and the safe application of engineered strains still has a long way to go.

With the continuous development of synthetic biology, engineered strains have showed great potential in various fields, however, more research is needed to ensure safety of its usage.

The Wind-up Cell system designed by iGEM Tianjin is designed to act as a universal tool to promote the safe application of bioengineered products in environments outside the laboratory, such as food, rivers, and the human body. In this project, we have constructed an engineered bacteria consisting of the Wind-up Cell system plasmid and the test paper detection plasmid, further exploring the feasibility of various applications of the Wind-up Cell system.

Figure 2. The potential application environments for engineered bacteria may include: a. Delivering drugs to specific locations within the human body. b. Breaking down environmental pollutants. c. Detecting toxic and harmful substances.

more details: Proof of Concept

Biological safety refers to the potential threats to the ecological environment and human health posed by the development and application of modern biotechnology, as well as the series of effective preventive and control measures taken to address these threats. Hewett et al.^[1] analyzed 44 risk issues associated with synthetic biology, categorizing them into risks to human health and risks to the environment. For humans, biosafety concerns may lead to allergies, antibiotic resistance, carcinogenicity, and pathogenicity. For the environment, it may result in environmental changes or degradation, competition with local species, horizontal gene transfer, and toxicity.

Our project is dedicated to developing a new tool to provide safety assurance for engineered microorganisms in a wide range of applicable environments. We call this tool the Wind-up Cell system, wherein it can inhibit and relieve the inhibition of toxic genes without altering the DNA sequence of the toxic genes through epigenetic modifications.

Figure 3. The principle of kill switch a. Epigenetic modifications suppress toxic genes without altering the DNA sequence. b. Epigenetic modifications are removed, leading to the expression of toxic genes.

Biological Conservation

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An important safety concern in synthetic biology is the intentional or unintentional release of synthetic organisms into the environment, leading to uncontrolled proliferation. Additionally, horizontal gene transfer is a common occurrence in nature.

Current common biological containment systems include active toxin-antitoxin systems, nucleases, and passive auxotrophic strains.

The Wind-up Cell system possesses the following characteristics:

The toxic gene is constantly in constitutive expression, with its sequence remaining unchanged. The toxic gene plasmid comprises a constitutive promoter and the toxic gene. Once the epigenetic modification inhibiting its expression is removed, cells are bound to die.

The engineered microorganisms with Wind-up Cell system do not require additional inducers to trigger the expression of toxic genes. Compared to common kill switches, this enhances safety in usage and reduces complexity, making it more user-friendly.

The engineered microorganisms with Wind-up Cell system can only function within the timeframe of delayed cell death. When the engineered microorganisms with Wind-up Cell system comes into use, they depart from the environment inducing epigenetic modification expression. The kill switch is then triggered, releasing the inhibition of the toxic gene, initiating a countdown to cell death. Therefore, the engineered microorganisms with Wind-up Cell system can only operate during this countdown, demonstrating their functionality exclusively during this countdown to cell death. This characteristic of initiating function after entering the death process also enhances the safety of synthetic biology applications.

Safety of Project Design

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To ensure the safety of the project, iGEM Tianjin has carefully designed the experimental plan and communicated thoroughly with the Safety and Security Program Officer.

Professor Weiwen Zhang from Tianjin University's Center for Biosafety Research and Strategy has proposed that our Wind-up Cell system should be capable of achieving an escape rate of less than 10^-8. He encourages the integration of epigenetic modifications with other methods to further enhance the lethality and precision of the Wind-up Cell system's regulation.

Figure 4. Professor Weiwen Zhang guided on the design of the Wind-up Cell system.

Wind-up Cell System

The Wind-up Cell system consists of a toxic gene plasmid and a tool plasmid.

MazF functions as a toxic Endoribonuclease to interfere with the function of cellular mRNAs by cleaving between A and C residues, which locate between the SD sequence and the initiation codon, leading to a halt in rapid cell growth and cell death^[2].

Figure 5. The principle of MazF

EcoRI is a restrictive endonuclease, which can specifically recognize GAATTC sequence and cut this sequence between G and A. The end point of the small segment after cutting is a sticky end protruding from the 5 'end. The expression of this gene can disrupt DNA sequences and cause cell death.

Figure 6. The principle of EcoRI

ccdB gene is a double-stranded DNA restriction endonuclease toxin, which achieves its toxic effects by encoding a protein called ccdB. The ccdB protein has the ability to inhibit DNA gyrase, which is an essential enzyme involved in DNA replication and transcription. By binding to DNA gyrase, the ccdB protein disrupts normal DNA synthesis and transcription, leading to inhibition of bacterial growth^[3].

The epigenetic modification suppression occurs exclusively in a specific environment to repress toxic gene expression. Once removed from this specific environment, the engineered microorganisms with Wind-up Cell system will unavoidably die. Within the epigenetic modification tool plasmid, there are inducible promoters and inhibitory elements. If outside the inducing environment, the inhibitory tool plasmid remains unexpressed, removing the inhibition on the constitutively expressed toxic gene, causing cell death.

In yeast, the engineered plasmid fuses dcas9 with a histone deacetylase enzyme. By using gRNA to specifically target the promoter of the toxic gene, the histone deacetylase enzyme induces the formation of heterochromatin, inhibiting the transcription of the toxic gene.

In E.coli, the toxic gene is suppressed through Dam methyltransferase methylating the promoter mioC and targeting the mioC promoter and the upstream open reading frame using the CRISPRi system.

Environmental Friendly

The yeast strain BY4742 we used in the experiments is a nutritionally deficient strain that is unlikely to survive in natural environments. E.coli MG1655 is a non-pathogenic bacterium commonly used in research and poses no harm to the environment itself.

The essence of the Wind-up Cell system is a kill switch, the ultimate effect of which is the death of engineered microorganisms possessing this system, preventing them from causing harm to the environment. The toxic gene expression products we chose are endogenous enzymes rather than chemical toxins, so they will not cause toxicity when released into the environment.

Figure 7. The Wind-up Cell system is environmentally friendly.

The safety of our project has been confirmed and approved through check-ins with the Safety and Security Program Officer.

Promotion of Biosafety Knowledge

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The realization of biosafety involves not only the development of biosafety-related technologies but also the improvement of public awareness of biosafety. During the project, iGEM Tianjin carried out various activities to publicize biosafety knowledge.

Community Promotion

We contacted the Yuhewan Community in Hongqiao District, Tianjin, and conducted public science education activities centered around the community. We designed three promotional brochures, which introduced the definition, significance, and relevant laws of biosafety , the issue of invasive alien species, and gene therapy respectively.

Figure 8. Community awareness newspaper

Debate Competition

In August 2023, we jointly organized an online debate competition with BIT-China team from Beijing Institute of Technology, with the theme "Promoting/Obstructing the Development of Scientific Research through the Formulation of Biosafety and Bioethical Regulations". We invited Professor Yi Wu, Professor Yingxiu Cao from Tianjin University, and Professor Bing Hu from Beijing Institute of Technology as judges, and there were many high school and university students as spectators.

Figure 9. Debate on biosafety and bioethics

more details: Human Practices

Laboratory Safety

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Safety is a priority in all experiments of our project. To minimize laboratory safety risks, team members received safety training before entering the laboratory. Every team member attached great importance to the safety requirements of laboratory work. Throughout the project, team members strictly adhered to the iGEM safety guidelines.

Specifically, the following points are observed:

1. Ethical Standards in Experimental Design: The suicide system we use effectively avoids environmental contamination by laboratory strains, and the entire project is harmless to humans. In the implementation of the project, we chose fluorescent proteins as the reporter gene, which allows testing without the addition of any harmful reagents.

2. Environmental Safety: After the plasmids we synthesized are transformed into Escherichia coli and yeast, the edited strains may cause environmental contamination. Therefore, the bacterial liquid used in the laboratory should be collected and disposed of after treatment to avoid microbial contamination of the environment.

3. Handling of Hazardous Reagents and Experimental Operations: When experiments need to be conducted under hazardous conditions, team members must comply with the requirements in the Tianjin Guidelines for Biosafety Behavior of Scientists and The Biosafety Law of the People's Republic of China. Hazardous reagents used (such as nucleic acid dyes) can have a certain impact on the health of experiment operators if exposed to the skin. Thus, team members must follow the requirements in the Material Safety Data Sheet (MSDS) during gel electrophoresis. Additionally, double-layered gloves should be used to prevent skin contact with toxic reagents during electrophoresis. When using ultraviolet light for equipment disinfection, we strictly follow laboratory standard procedures to avoid direct exposure of the skin to ultraviolet light.

4. Safe use of experimental equipment before starting an experiment: carefully read the instructions, familiarize oneself with equipment operation, and avoid accidents caused by unfamiliarity with the operation.

In addition, we have a responsible teacher for each laboratory to manage our safety affairs, and we have college and school-level institutions as supervisors for laboratory safety.