Safety Introduction

Overview

As we all know, safety is the paramount factor which we must consider seriously. We strictly follow the safety requirements of iGEM and comply with the relevant laboratory safety regulations.

Furthermore, we identified potential risks in advance, both in the experiments and in future applications, proposing relevant countermeasures. Prior to commencing any experiments, we conducted comprehensive safety training and skill development session for all team members. Throughout the entire experimental process, we consistently followed establishedsafety guidelines and the legal protocols to foster a secure working environment. Additionally, we dynamically adjust procedure as necessary and maintained a stage summary of issues to ensure overall experiment safety.

Laboratory safety

Our laboratory operates at BSL-II, adhering to the General Biosafety Standard for laboratories handling pathogenic bacteria in the People's Republic of China. We implement fundamental safety protocols, which include requiring all personnel to wear long pants, closed-toe shoes, lab coats, and gloves while working in the lab. Protective goggles are also mandated when necessary. Additionally, all sterile procedures are performed within a Biosafety Cabinet to ensure a controlled environment. Furthermore, there is a strict separation between the experimental area and the office space to minimize contamination risks.

The main safety features of our laboratory include:

1.Some common safety facilities and emergency devices

2.Biological safety cabinets

3.The classified garbage bucket: the black garbage can mainly contains unpolluted garbage (e.g., sealing film), and the yellow garbage bucket contains biochemical drugs and other wastes such as culture medium, and test tubes.

4.Sufficient personal protective equipment and a counter for storage.

5.Mechanical ventilation system

6.Entry and exit registration

7.Safety assurance documents: laboratory safety codes, laboratory procedures manuals, etc.

Personal Safety

Biosynthetic research carries inherent risks due to its nature. To ensure a safe working environment for both the team and the progress of the project, we have implemented various safety protocols in our laboratory work. Prior to commencing experiments, we conduct comprehensive safety training and skill development for every team member. Throughout the experimental process, we meticulously document all operations performed, with faculty members supervising project safety. After each experimental phase, we compile a summary to evaluate and enhance safety measures, ensuring that the overall experiment is conducted as safely as possible.

1. Personal protection training

Through our laboratory safety training, we ensured operational safety, preventing any leakage of engineered bacteria containing antibiotic resistance genes.

Prior to the commencement of the practical project, team members underwent three months of laboratory safety training. In alignment with the safety protocols established by the CUG Lab, comprehensive safety instructions were provided before the project began and were strictly adhered to throughout. Each team member was equipped with protective gear, including a lab coat, mask, and disposable gloves, to maintain a safe working environment.

2.Supervisors to support us in managing risks

In our laboratory, safety is overseen by two designated teachers, Zhou Jiang. If we identify any hazards or risks during our project, we promptly consult with Zhou Jiang, and our supervisors for assistance. Additionally, we ensure that all safety concerns are communicated to the laboratory director, Huanying Pan, for further confirmation and guidance.

Risk identification:

In our project, heavy metals were utilized to confirm gene specificity, which can pose risks to human health and the environment. Irregular handling of these materials may lead to chronic poisoning due to accumulation in the body. Therefore, if anyone accidentally comes into contact with hazardous organisms or chemicals, or if an engineered organism is inadvertently released, it could compromise safety and pose significant risks.

Management:

To address these challenges, we provided comprehensive safety training and experimental skill development for all team members prior to the experiment. During the experimental process, we meticulously documented each operation and had supervisors monitoring safety protocols. After completing each stage, we conducted summaries to enhance overall safety.

Furthermore, looking ahead, the application of engineered bacteria holds potential for biomining in outer space. Specifically, we propose transporting A. ferrooxidans to a space station for this purpose, facilitating gold extraction through engineering techniques. This approach offers an environmentally friendly solution that minimizes pollution, improves metal recovery, and addresses the limitations associated with current tailings disposal methods.

So we use these measures:

1. Accident reporting
A robust accident reporting system is essential to our operations. We have instituted a protocol requiring that any exposure to hazardous materials or incidents involving genetically engineered organisms be reported immediately. Each incident must be documented and assessed to facilitate the initiation of appropriate corrective measures. This ensures that we maintain a safe working environment and continuously improve our safety practices.

2. Wearing protective goggles, etc.

3. Inventory controls
Strict inventory management is enforced to monitor the use and storage of all hazardous substances and engineered organisms. Regular audits ensure accountability and prevent unauthorized access or misuse.

4. Physical access controls
Limited access to laboratory areas containing hazardous materials or genetically modified organisms is strictly regulated. Access is granted only to authorized personnel, and all entries and exits are logged.

5. Data access controls Sensitive data regarding our genetically engineered bacteria and experimental procedures are protected with secure access protocols. Access is restricted to personnel on a need-to-know basis, safeguarding our research.

6. Lone Worker or Out of Hours policy A policy is in place to ensure the safety of individuals working alone or outside regular hours. This includes designated check-in procedures, emergency contact information, and panic alarm systems.

7. Medical surveillance Regular health check-ups are mandatory for all team members working with hazardous materials or engineered organisms. This proactive measure monitors the health of our team and provides immediate response in case of exposure.

8. Waste management system

Project design safety

Strain selection and organisms selection:

The Acidithiobacillus ferrooxidans strain we selected was obtained from ATCC, and it is suitable for metal bioleaching under conditions of strong acidity and heavy metals. We explored more efficient modules of c-di-GMP and dynamically optimized the construction of synthesis modules to enhance bioleaching efficiency and biofilm formation. Specifically, we will utilize Escherichia coli SM10, DH5-alpha, and BL21.

Additionally, we employed sulfuric acid to adjust the pH levels. For heavy metal induction, we used chloroauric acid, chromium chloride, zinc chloride, copper sulfate, and nickel chloride. It is important to note that these substances may have implications for environmental safety and personal health.

Management:

To neutralize reactions with sulfuric acid in wastewater, we add alkaline substances such as sodium hydroxide and limestone.

For chloroauric acid, we ensure thorough cleaning of equipment post-operation and require the use of protective gloves and masks. We take precautions to prevent its release into the environment, and after use, we employ a reducing agent to convert it to gold.

Regarding chromium chloride, we avoid contact with skin and eyes, precipitating it by adding calcium salts.

For zinc chloride, personnel must wear chemical safety goggles, protective clothing made of corrosion-resistant materials, and rubber gloves. In cases of dust exposure, gas masks are mandatory. After use, we add sulfides to initiate a precipitation reaction before disposal.

With copper sulfate, closed operations with adequate local exhaust ventilation are essential. Operators must be specially trained and adhere strictly to standard operating procedures. It is recommended that they wear self-priming filter dust masks, chemical safety goggles, poison-proof protective clothing, and rubber gloves, while minimizing dust generation and avoiding contact with acids and bases.

For nickel chloride, appropriate gloves are required, and it must not be discharged into drains. After use, we add sodium hydroxide or calcium hydroxide to facilitate a precipitation reaction prior to disposal.

Project to manage hazard:

The Acidithiobacillus ferrooxidans strain we modified was obtained from ATCC. We designed a plasmid and successfully transferred it into E. coli SM10, utilizing this system in conjunction with A. ferrooxidans to enhance biomining efficiency. Furthermore, we developed a genetic killing switch employing the mazF gene to ensure that A. ferrooxidans survives exclusively in our target environment. This approach allows for controlled survival and activity of the modified strain under specific conditions, optimizing its application in biomining processes.

We synthesized the mazF gene in our killing switch, which encodes the toxin protein in MazE-MazF, toxin-antitoxin module, functioning as an mRNA endonuclease.

Cycle 4

To address the potential ecological risks of releasing genetically modified organisms into the environment, we are developing a kill switch. This safety feature is designed to ensure that the engineered bacteria only survive under specific conditions, particularly in the presence of Au ³⁺ ions. To implement this, we have integrated a classical inverter gene circuit, which includes the cI repressor from the E.coli λ phage and its associated promoter P_R, into the gold-binding gene circuit. Additionally, we have introduced the toxic protein MazF, creating a system that ensures the engineered bacteria can only thrive in environments rich in Au³⁺.

The construction of the suicide gene circuit involved a double enzyme digestion method. We amplified the cI-PR segment, adding restriction sites via PCR. The cI-P_R gene segment and the vector pYDT-golS-P_gol were then digested with restriction enzymes to produce compatible sticky ends, which were subsequently ligated to create the expression vector pYDT-golS-P_gol-cI-P_R.

For the integration of the mazF gene, which encodes the toxic protein, we initially optimized its codon usage to match that of A. ferrooxidans. The codon-optimized mazF gene fragment was synthesized by Tsingke Biotech Co., Ltd. Following this, we employed the double enzyme digestion method to create sticky ends on the mazF gene and the vector, which were then ligated to form the final expression vector pYDT-golS-P_gols-cI-P_R-mazF. This comprehensive design ensures that our engineered bacteria are not only equipped for the task of gold recovery but also safeguarded with a built-in mechanism to contain their survival to controlled conditions.