. Safety .
1.Overview
Every member of the team knows and pays attention to the safety of every step of the competition, understands the inherent risks in the process of working, and invests a lot of energy in preventive measures. Everyone believes that working in a safe and secure environment and working together in a safe and comfortable environment is an essential part of responsible innovation, research and collaborative communication. The team has done full and complete safety work in the aspects of preventive measures, actual operation and post-guarantee. By continuously identifying and managing potential safety risks, the team minimizes harm to the iGEM team, stakeholders, project participants, society, and the environment. Therefore, we considered the overall situation in terms of security from physical and laboratory related risks to the privacy security of project participants. This page will discuss the security measures we have taken in every conceivable way.
2.Safety of experiment
2.1 Laboratory safety
All experiments performed by the HuBu-4-CHN team were in compliance with the 2024 iGEM safety policy and rules. Most organisms and parts used or generated are on the iGEM 2024 whitelist. Parts that are not on the whitelist are successfully submitted via the check-in form before use. Throughout the development process, all national and institutional rules and regulations were strictly followed.
2.2 preparation before laboratory work
Because this year's iGEM team was partially working in ML-1 and ML-2 classification laboratories (equivalent to biosafety levels 1 and 2, but only performing ML-1 work), all team members had to attend multiple introductory sessions conducted by laboratory technicians from each laboratory. These presentations detail safety policies and rules within specific laboratories. The location of all safety equipment is shown in this introduction (Figure 1).
- Figure 1| Safety supplies in the laboratory. The left side is the emergency spray equipment, the upper right corner is the use of precautions, the lower right corner is the fire extinguisher.
- Figure 2| The students in the experimental group, wearing experimental clothes, masks and gloves, were doing the experiment in a standardized way.
2.3 Safety measures during laboratory work
| Lifetime | Safety Precautions |
|---|---|
| Personal Protection | It is required to wear protective clothing and all-inclusive leather shoes when entering the laboratory. Long hair must be tied back. When it comes to reagents with safety risks such as acid, alkali, alcohol, and corrosive agents, it is required to wear goggles and gloves. Clean your hands with disinfectant before leaving the laboratory. |
| Security Device | The safety preparation page before laboratory work shows the functional areas of safety facilities such as fire extinguishers and emergency sprinklers. |
| Environmental Safety | The building is equipped with escape routes, escape plans and security staff studios. |
| Chemical Safety | The laboratory was kept clean before and after the experiment, and the chemicals were classified and stored independently and safely. |
| Waste Treatment | The waste liquid will be dumped into the waste liquid tank before being properly disposed of. |
3.Project Design Safety
3.1 intestinal probiotics as the base cells
We chose Escherichia coli Nissle 1917 (EcN), a non-pathogenic probiotic bacterium that naturally occurs in the human gut and is classified as biological safety level 1 (BSL-1), as the backbone cell. EcN is mainly used clinically to treat inflammatory gastrointestinal disorders such as Crohn's disease and ulcerative colitis. It colonizes the gut to prevent pathogenic bacteria from invading, protects and repairs the intestinal mucosal barrier, and participates in host immune regulation by balancing the secretion of immune factors and enhancing immune function to alleviate inflammation. Recent studies have found that EcN has tumor-targeting effects, and when used in combination with chemotherapy drugs, it can enhance anti-tumor effects. All laboratory work involving genetically modified bacteria has been recorded in the transgenic notebook.
3.2 Safe probiotic-secreted outer membrane vesicles were selected as delivery vehicles
Bacterial outer membrane vesicles (OMVs) secreted by Escherichia coli Nissle 1917 (EcN) were used as the Cas9 RNP delivery system. OMVs are small nano-liposomes released by Gram-negative bacteria, typically ranging in size from 20 to 200 nm. They do not function as intact bacteria and therefore do not cause bacteremia when they function in vivo, reducing the risk of bacterial infection or disease transmission while reducing the immune response. The advantage of OMVs as a delivery system is that it avoids the risk of transmission of antibiotic resistance genes and gene transfer that may be brought by live bacteria. OMVs are less toxic to cells and provide a higher safety profile compared to conventional adenoviral vectors and cationic liposomes.
3.3 Proof of Concept Experiment
For safety reasons, we chose not to conduct animal experiments, but to validate the efficacy of the OMVS-targeted delivery CRISPR/Cas9 gene editing system in commercial HeLa cell line.
3.4 gRNA registration forms targeting the human genome
Our project strictly follows the iGEM security policy, and for project components not on the whitelist, we have submitted the whitelist Check-in Form for risk management. The OMVs delivery system will be validated against the HSP70 gene in HeLa cell line. Since grnas targeting human genes are not included in the whitelist, we deeply understand the importance of safety risk management in synthetic biology applications and strictly abide by iGEM safety rules to ensure the safe progress of the project.
3.5 Potential risk assessment
In the future, OMV-Cas9 RNP delivery system is expected to be used for the treatment of human genetic diseases. Gene editing to treat disease while maintaining normal cellular function is achieved by delivering the system to the target site by intravenous injection. Our team and discussions with professionals have shown that the OMVs gene editing delivery system has potential risks in practical application. If CRISPR gRNA misrecognizes non-target DNA sequences, it may interfere with the function of healthy cells and even lead to cell death. Therefore, the off-target effects of gRNA must be rigorously tested before cell line experiments.
3.6 The immunogenic side effects of Cas9 RNP were further reduced by gene mining and synthetic biology
In the future, our project plans to introduce a two-plasmid expression system of Cas9 RNP and alkaline phosphatase (ALP), aiming to reduce the immune response of gRNA in humans. We planned to co-transform the ALP expression plasmid from E. coli with the expression plasmid of Cas9 RNP into EcN cells. ALP is able to catalyze the removal of the 5'-PPP modification of gRNA to generate 5'-OH to reduce the side effects of stimulating the innate immune RIG-I pathway. Given the presence of pre-existing antibodies against SpCas9 and SaCas9 proteins in more than 70% of humans, we will also explore the use of genetic engineering and selection in extreme environments to obtain Cas9 RNPS that elicited lower immune responses in humans. This strategy aims to reduce the immunological risks of gene editing and improve the safety and efficacy of therapy.
4.Human practice safety
The security of the people is the supreme core value of national security. As a qualified iGEM team, we attach great importance to the study of privacy responsibility for project participants. We systematically studied the contents related to privacy security in the law of the People's Republic of China, including the Constitution of the People's Republic of China (revised in 2018), the Civil Code of the People's Republic of China (2020), and the Decision of the Standing Committee of the National People's Congress on Strengthening the Protection of Network Information (2013). Together, these laws, regulations and policy documents guarantee the right to privacy of citizens of the People's Republic of China. After consulting the Legal Aid Center of the People's Republic of China, the privacy protection of project participants was effectively approved, and the human practice research involving project participants was formally carried out. We prepare the privacy protection notice with the help of the legal advice hotline, so as to make our project consultation interaction publicly comply with the laws and regulations of the People's Republic of China and effectively protect the project participants.
We always believe that building a good relationship of trust with our project participants is beneficial to the development of both parties. We infected the participants with warm and sincere words and shared our project with them. In the process, we not only build a friendly relationship, but also make them feel like an integral part of our project. Here, you can swipe below to see the privacy protection notice for project participants as an example. This includes information we collect about program participants, how we process the information, and how we securely store and use the information to ensure the privacy and security of program participants.
5.Safety Campaign
5.1 The 1st International Symposium on the Application and Safety of Escherichia coli Nissle 1917
Objective:The first International Symposium on the Application and Safety of Escherichia coli Nissle 1917 was organized by the School of
Biological and Pharmaceutical Engineering of Nanjing University of Technology and Peking University Health Science Center.
Implementation plan: Our team was invited to participate in the first "International Symposium on the Application and Security of
Escherichia coli Nissle 1917", which was held in the form of an online Tencent meeting.
Details:
1. Discuss the potential application and safety issues of Escherichia coli Nissle
1917 in the field of synthetic biology with the participating iGEM team in the online Tencent meeting
2. After the meeting, the division of labor and cooperation will complete the writing of the EcN white paper
Attached:
5.2 Compilation of the "Ethics Manual of Synthetic Biology"
Purpose: Through jointly writing the "Ethics Manual of Synthetic Biology" with various universities and based on the project of our team,
we aim to supplement relevant safety issues, establish the ethical framework of synthetic biology, promote the healthy development of
synthetic biology technology, ensure that it adheres to ethical principles in the application process, and facilitate the improvement and
development of the research and practice of the team project in the directions of safety, responsibility, and in-depth education.
Implementation Plan: Multiple iGEM teams, such as CJUH-JLU-China and our team, collaborate to jointly
write the "Ethics of Synthetic Biology" manual.
Activity Content: Hosted by the relevant iGEM team of Jilin University, through online communication, discussion, and division of labor
to write the ethics manual of synthetic biology,
and share relevant track experiences.
Details: On August 11, 2024, our team's HP began working on the initial draft of the manual.CJUH-JLU-China serves as the chief editor,
completing the writing of the preface, integration and typesetting, text review, and other contents. Each team writes the responsible
part separately and submits the completed draft to the CJUH-JLU-China team.
After each team makes phased progress, an online meeting will be held for summary.
Based on the projects of each team, ethical topics of synthetic biology are explored to provide an ethical guidance framework for synthetic biology researchers, policymakers, ethics review institutions, and the general public in China and even globally, guiding synthetic biology research and practice towards a more responsible and sustainable direction.
Our team (HuBu-4-CHN) is mainly responsible for writing the first draft of the ethics manual of synthetic biology. Based on our
team's iGEM project, we are mainly responsible for writing the manual in the medical aspect.
Attached:
