Laboratory Safety
Attendance of Biosafety Training
At our university, it was mandatory to attend biosafety training before using the laboratory. Therefore, all Wet Team members attended the biosafety training and received permission to conduct experiments.
Attendance of Biorisk Management Training
We conducted biorisk management training for students with Mr. Iki from the National Institute of Infectious Diseases. This training included not only lectures on biosafety and biosecurity but also group discussions to foster the ability to anticipate risks and develop strategies to prevent them.
In the morning, we learned about what risks are, the possible risks that might arise when starting an experiment, and past incidents. In the afternoon, we used LEGO bricks to represent the risks we envisioned and communicated our ideas. It also provided an opportunity to think about risks from perspectives we hadn’t considered through discussions with others. Based on the content of this training, the participating Wet Team members gained the "risk management skills" necessary for conducting synthetic biology experiments.
【Training content】
・Lecture on Biorisk Concepts
First, we received a lecture on the concept of biorisks. In this lecture, the concept of biorisks was explained from both a biosafety and a biosecurity perspective.
Biosafety:
Biosafety is about measures to protect people and the environment from the risks posed by pathogens. This includes the "awareness" of not harming oneself or others, the "attitude" of following rules and manuals, and the "actions" of appropriate physical containment in the lab. These fundamental concepts ensure the safe handling of microorganisms and accurate experimental results, helping prevent major accidents.
Biosecurity:
Biosecurity is about measures to protect pathogens from misuse by malicious individuals or inappropriate actions.
Specific risks include zoonotic diseases, biological weapons, invasive species, and food poisoning. From a biosecurity perspective, risk assessments, consideration of potential threats from emerging technologies, and strengthening of laws and regulations can prevent incidents or accidents.
Based on the concepts of biosafety and biosecurity and their examples, we learned about the risks that could arise during experiments and what precautions we should take. This knowledge was shared among the Wet Team members, enhancing the safety of our activities in synthetic biology experiments.
・Biorisk Imaginary Training with LEGO Bricks
Next, we conducted Biorisk Imaginary Training using LEGO bricks in a group setting. We created two types of LEGO models to represent our ideas about safety and security.
The first model was titled "Researchers Threatening Biosafety and Security." Each participant chose a sample model to recreate and then added one LEGO piece to represent a researcher who threatens biosafety and security. One Wet Team member created a model showing how a genetically modified organism they made unknowingly harmed them. Even among participants who chose the same model, different perspectives resulted in different creations, allowing us to gain a broader view of safety and security.
The second model had the theme "Express Your Thoughts on Biosafety and Biosecurity Using LEGO." After creating their own models, each participant presented and discussed their creation with the group. One Wet Team member created a model titled "An Experiment Classroom Run by Amateurs," representing an experimental classroom that had been opened without considering biosafety or security.
Fig.1 Photo of LEGO bricks created
Using LEGO bricks to express our thoughts allowed us to visually convey ideas that might be challenging to verbalize, and others provided insights we hadn't previously considered. Seeing others’ creations also gave us a glimpse into their perspectives.
・Practical Biorisk Management (Group Discussion)
Following this, we practiced biorisk management. Biorisk management involves analyzing methods and developing strategies to minimize the potential for biorisks to occur. We simulated this practice through group discussions using distributed cards and worksheets.
In one exercise, participants were given cards describing the characteristics of two microorganisms, A and B, and asked to consider the risks and benefits of creating recombinant organisms A'B and B'A by introducing genes from one into the other. Through this group discussion, we concluded that creating B'A could involve unexpected risks not present in creating A'B.
Another exercise involved cards describing scenarios with insufficient biosafety considerations. We evaluated potential risks, including how incidents like exposure or leakage might occur, factors influencing the likelihood of incidents, the importance of initial risk assessments, and the need for additional risk measures.
Based on these simulated exercises, we felt the importance of thoroughly researching the surrounding information about microorganisms to avoid unexpected risks when conducting synthetic biology experiments.
Fig.2 Photo with Mr. Igi of the National Institute of Infectious Diseases
Laboratory Environment and Equipment
The lab is equipped with a Class II safety cabinet and a clean bench.
Fig.3 Clean benches and safety cabinets
The lab also has an autoclave for sterilization.
Fig.4 Autoclave
Before conducting experiments in the lab, we explain the procedures to lab members (PI, PD, graduate students) and proceed with their approval. Additionally, we learned how to use experimental tools and equipment from graduate students and professors before using them.
The laboratory is classified as Biosafety Level 1, and we only handle non-pathogenic microorganisms that belong to a whitelist.
Project Design
From a biosafety perspective, a kill switch mechanism was devised to prevent genetically modified organisms from surviving outside the culture medium. Since tyrosinase activity requires copper ions, we focused on adding CuSO4 to the medium and used copper ions as the inducer for the kill switch.
CueR binds with reduced Cu+ within the cell, which activates the copA promoter. This leads to the production of TetR, which binds to the tetracycline operon operator and represses transcription, preventing the production of a cytotoxin. However, outside of the medium, the copA promoter is suppressed, allowing the tet promoter to activate and produce the toxin, causing cell death. The toxins used were holin and endolysin.
Research on holin and endolysin has provided insights into their safety when used for therapeutic purposes. For example, Schmelcher (2012) found that endolysins primarily target bacterial cell walls and do not affect eukaryotic cells, making them generally safe for therapeutic use. However, when treating Gram-negative bacteria, the use of outer membrane proteins (OMPs) or other enhancers may pose potential safety concerns, as they could also interact with eukaryotic cell membranes, necessitating careful evaluation before clinical application. Moreover, a study involving the use of phage-derived endolysin for treating skin infections reported no adverse effects, suggesting that endolysin can be safely applied topically against Gram-positive bacteria.
Fig.5 Design of Kill Switch Parts
This kill switch design can be adapted for other projects using copper ions as a medium component.
kill Switch Model
We created the kill switch model that based on parts, and tested.
The result is shown below.
Fig.6 the result of kill switch model
This figure shows that the production of holin increases as the copper ion concentration decreases.
The threshold concentration of Holin was set at 5e-7M (more detail in Model page). This Holin concentrations induce cell death. When the copper ion concentration is below 1e-10M, its steady state is greater than the threshold concentration of Holin. The kill switch is turned on after about 10 minutes when the copper ion concentration is below 1e-10M in the environment surrounding the cell drops.
Our designed kill switch can turn the switch on and off in a copper ion concentration-dependent manner. When the medium contains copper ions at a concentration of 1e-6M, the kill switch is off. However, when the bacteria leave the medium and the copper ion concentration around the bacteria falls below 1e-10M, the kill switch is clearly on.
