Overview Our project focuses on the use of genetically modified adipocytes for detection of breast cancer. Safety is a fundamental aspect of our project. We recognize that synthetic biology involves working with biological materials and systems that require careful handling and consideration to prevent any potential harm to ourselves, the environment, and the broader community. Throughout our project, we have adhered to the highest standards of safety, following iGEM’s safety policies and guidelines. We have also identified potential risks in advance and implemented necessary precautions to ensure that our project is conducted responsibly and securely. Self-Check List
Self-Check Entries Answers
Did the team make a contribution to biosafety and/or biosecurity?
Yes, we have considered how to ensure that the secretions of engineered cells and their secretion processes do not negatively affect normal physiological processes.
Is their contribution well-characterized and/or well-validated?
Yes, we have validated the activity of adipocytes through CCK-8 experiment, which is shown in our page “results”. We also developed BEAM to assess the ethical risks of the project, which is shown in our page “model”.
Did the team build upon existing knowledge, understanding, tools or approaches?
Yes, we have reviewed extensive literature and previous iGEM projects, which our design is based on.
In addition to broader safety work, has the team managed risks from their project appropriately?
Yes, we have managed risks from our project appropriately by conducting regular risk assessments and providing effective responses.
Has the team addressed the use of synthetic biology in the real world?
Yes, we have taken into account the risks that may arise in the application of the product and have carried out an ethical analysis by the method of Bio-Ethos Assessment Model (BEAM).
Lab Safety General lab safety We conducted our wet lab work at the Students' Laboratory in the China-Japan Union Hospital, Jilin University, which is qualified for working with Risk Group 2 organisms. All work conducted in these spaces and with the available equipment was fully compliant with international regulations. We ensured that all activities adhered strictly to China national biosafety regulations. Every plasmid created and bacterial strain used was meticulously documented in a notebook, with a hard copy stored in the laboratory. Furthermore, we implemented comprehensive safety measures and effective risk mitigation strategies to minimize potential hazards.
experimental equipment
Waste is classified into household waste and medical waste. The medical waste is regularly sent to specialized facilities for disposal so that waste chemicals, bacteria, and other substances can be safely disposed of without harming the environment or the experimenter. Personal lab safety Before conducting experiments, all members of our wet lab receive mandatory laboratory safety training. The experimenters are provided with lab coats and gloves, and work in pairs to minimize potential accidents. Daily experiments are meticulously recorded to ensure both safety and accuracy.
protective tools and safety rules
Biosafety In our experiments, we used Human embryonic kidney cells HEK-293T, Human preadipocytes, Escherichia coli DH5-alpha, recombinant Adeno-associated dependoparvovirus A(rAAV9), and human breast carcinoma cells MDA-MB-231. These organisms and cells all come from reliable suppliers, and the cells have been identified and certified to be free of known pathogens. In addition, we also placed great importance on waste management and strictly adhered to “Biosecurity Law of the People's Republic of China”. All waste was placed in yellow biohazard bags and was marked and disposed of by specialized agencies. The engineered bacteria and cells used in our experiments were carefully handled within the laboratory to prevent them from leaking into nature. Furthermore, our cell experiments were conducted under the guidance of experienced laboratory personnel such as Instructor Peipei An, and we followed good laboratory practice and wore personal protective equipment (gloves, lab coats, etc.) during all experiments. Project Design Safety About our project Our project aims to provide breast cancer patients with more stable and long-term prognostic monitoring. Currently, ultrasonography and other imaging modalities have proven to be effective tools for assessing breast health in clinical settings, but there is a lack of a diagnostic modality that can monitor the inside of the breast in real time without any imaging tests. With the progress of research, it has been found that normal adipocytes are gradually "reprogrammed" to become cancer-associated adipocytes after contact with tumor cells. When stimulated by cancer cells, adipocytes secrete a variety of characteristic cytokines, proteases and other small molecules. Our project is based on the principle of adenosine deaminases acting on RNA(ADARs) , which allows us to perform detection of target RNA molecules with specific sequences. By detecting genes specifically upregulated in cancer-associated adipocytes (CAAs), we can assess the recurrence of breast cancer. Output: Gluc In our project design, the indication method for breast cancer is as follows: engineered adipocytes secrete Gaussia Luciferase (Gluc) after contacting with the trigger, which enters the urine through kidney metabolism. By adding its substrate, coelenterazine, to the urine sample, a light signal visible in the dark can be generated. For engineered cells implanted in normal human tissues, it is undoubtedly crucial to ensure that their secretions and secretion processes do not affect normal physiological functions. Firstly, the humanized form of Gaussia luciferase effectively expresses in mammalian cells. Biochemical studies shows that the expression of this protein in cells can be achieved by transfecting plasmids encoding the humanized form of Gluc into cells. Existing research has successfully achieved its expression in human fibroblasts. Secondly, Gluc, as a secretory reporter protein, can be secreted by mammalian cells into the circulatory system and enter the urine through kidney metabolism. Adding its substrate, coelenterazine, to urine samples can produce a light signal visible in the dark. Previous experimental results shows that after expressing Gluc in tissues of mice, the Gluc enzyme activity in urine significantly increases. These studies support our design of using the light signal emitted from the reaction of Gluc in urine with its substrate as a diagnostic output indicator. Thirdly, Gluc, as a widely used secretory luciferase in biomedical research, has high biosafety. Previous studies shows that Gluc has not exhibited significant cytotoxicity, irritancy, or immunogenicity in human body, making it a suitable reporter protein. HP/Education Safety In promoting the human practice (HP) and education (Edu) activities of the ABCS project, we built a safety management system that covers the entire process of activities, ensuring that each interaction takes into account scientific nature, safety, and humanistic care. Activity Safety In order to avoid security risks during HP/Edu activities, the team has established a dual mechanism for safety management: 'Safety Notification - Process Control'. Before the start of any activity, a “Safety Responsibility Notice for ABCS Project Activities” was distributed to participants (including community residents, patients, students, etc.) to clarify the purpose, process, and safety precautions of the activity, with team members guiding throughout the entire activity.
safety responsibility notice
Regarding activities such as interviews with patients that involve sensitive groups, in addition to physical safety protection, we paid extra attention to the psychological safety of participants—We trained the interviewers to use “empathetic communication” in advance, avoided mentioning traumatic topics that may cause discomfort to patients, and clearly stated in the notice that 'participants have the right to suspend/terminate the interview at any time', ensuring that the activities safety covered both physical and psychological perspectives. Personal Privacy Safety Considering the specificity of the ABCS project that it involves patients’ medical information, the team established strict privacy safety management procedures: before conducting patient interviews, one should submit an ethical review application to China-Japan Union Hospital of Jilin University, obtain a “Ethics Approval Document of Research on Human Subjects” from the hospital's ethics committee, and clarify that the interview process and the scope of information usage comply with medical privacy protection regulations. When we collected basic information and interview contents of patients, all interviewed patients have signed the “Informed Consent Form”. We clearly informed them of the purpose of the records and that all obtained information would only be used for the development and analysis of the ABCS project, without disclosing it to any third parties, ensuring that privacy protection ran throughout the entire process of information collection and use. Ethical Safety We actively interviewed experts in the fields of ethics, philosophy, social sciences, law, and medicine regarding the ethical issues involved in the ABCS project to obtain professional advice, develop risk response strategies, and avoid the lack of adaptability caused by a single ethical standard.
interviews
In response to the gap of lacking quantitative tools in existing ethical assessments, we originally developed the “Bio-Ethos Assessment Model (BEAM)”. This model combines structural equation modeling and Bayesian network analysis to quantify ethical risks from various dimensions, providing a basis for ethical decision making and achieving an upgrade from “qualitative judgment” to “quantitative assessment”. Social Responsibility When determining the output signals of the detection system, we considered the “skin melanin synthesis” plan — engineered adipocytes cause the skin to produce melanin markers after sensing breast cancer signals. However, after assessment by Dr. Yang Bin, a breast surgeon, and team discussions, we found that this plan had significant risks—permanent surface markers could exacerbate patient anxiety and might lead the public to misunderstand that “synthetic biology can casually change the appearance of human body”, causing social panic about “technology misuse”. Therefore, we decisively rejected this path and chose a gentler, non-invasive output method of “luciferase” in urine, ensuring the convenience of detection while avoiding causing negative social cognition. Policy Analysis iGEM policies iGEM emphasizes that safety and responsibility are core issues that must be addressed in scientific research. Since biological experiments carry certain risks, we strictly adhere to all iGEM rules and policies. Our priority is to select biological materials listed on the White List, and any usage of materials not on the white list was checked-in prior to experiments. We are fully aware of the 4 Risk Groups and have ensured not using any organisms or their parts classified in Risk Groups 3 and 4. We have also ensured that no release beyond containment occurs throughout the stages of our experiments, and all waste has been reasonably managed by the lab directors. Furthermore, we have not tested our product on humans. JLU official policies on laboratory safety We conducted all our lab activities under the university lab safety management regulations. All students at Jilin University with laboratory courses are required to take a compulsory course on laboratory safety in their first semester and must pass the exam before they can enter the lab to conduct experiments. This course covers topics such as the classification of hazardous reagents, risks associated with commonly used chemicals, firefighting measures, personal protective measures, standard experimental procedures, laboratory regulations and so on. Therefore when conducting experiments, we are able to strictly adhere to the laboratory safety regulations and the college's laboratory safety management protocols, ensuring bio-safety and the safety of all personnel. National regulations Ultimately, our experiment strictly follows the national regulation Measures for the Safety Management of Genetic Engineering. Regarding Articles 6 and 8, our project is classified as Safety Level 1, posing no risk to human health or the environment. Regarding Article 9, through discussions with our advisor, we determined the vectors and hosts used in the project and confirmed that both our researchers and laboratory are capable of mitigating potential hazards. Regarding Articles 13, 14, and 15, our project was submitted to the college for review and received approval from the college's administrative authority. Regarding Articles 19, 20, and 21, we strictly adhere to laboratory safety regulations. The disposal of laboratory waste is managed by the lab supervisor in collaboration with professional biochemical waste treatment agencies. All our researchers have completed comprehensive emergency response training and possess standard experimental techniques. Regarding Article 24, we maintain detailed records of laboratory usage and safety supervision in a logbook, which is properly kept.