Overview

Our team is well aware that responsible innovation is the cornerstone of synthetic biology research. To this end, we have developed a comprehensive safety assurance strategy that runs through the entire project, is multi-dimensional and in-depth. This strategy strictly adheres to the iGEM competition regulations and national and institutional biosafety laws and regulations, and based on the principle of "prevention first, control foremost", systematically integrates safety management into the entire process from molecular design, strain construction, experimental operation to waste disposal. At the strategic level, we have endowed engineered organisms with built-in safety attributes through the safe selection of chassis organisms, multiple physical and genetic leak-proof mechanisms, and inducible biocontainment systems. At the operational level, we rely on standardized experimental facilities, well-trained research teams, and complete institutional procedures to ensure that every operational link is strictly controlled. This section will elaborate in detail on the specific measures and design concepts we have adopted to achieve the goal of "absolute safety and full compliance".

Built-in Safety mechanism

Qualifications

This project strictly adheres to all the rules and policies of iGEM and resolutely prohibits any activities explicitly forbidden by iGEM. We have a clear and thorough understanding of the iGEM whitelist. All organisms, parts, and related operations involved in the experiment are within the scope of the whitelist.

Compliance

All the experimental contents of this study strictly comply with the relevant national laws and regulations as well as industry norms. During the design of the experimental plan, the execution of experimental operations, and all other links, compliance reviews have been conducted to ensure that there are no operations that violate legal provisions or exceed the permitted scope. It fully meets the legal and compliant standards for scientific research. During the information collection process, we also follow the laws and regulations as the fundamental basis and carry out work strictly in accordance with the legal procedures and authorities. The objects, scope, and methods of information collection all comply with the legal requirements for data security and personal information protection, and there is no illegal collection or forced acquisition of information. We guarantee that the information collected will be used solely for research purposes and strictly ensure the confidentiality of the information. We will never disclose it to any organization, unit or individual.

Rationale

During this preclinical research stage, we chose to use the highly standardized laboratory yeast strain INVSc1 as the model vector to build the engineered yeast. We selected INVSc1 as the research tool based on its high homology in core biology with the target clinical application strain - Saccharomyces boulardii (also taking into account the objective factors of the laboratory's technical conditions). INVSc1 is a derivative strain of Saccharomyces cerevisiae, and whole-genome sequencing indicates that S. boulardii is extremely similar in genetic background to S. cerevisiae, and can be regarded as a special probiotic variety of the latter. Previous studies have shown that the core principles successfully verified in INVSc1 can be replicated in S. boulardii. Both share the same protein synthesis, secretion and metabolic basic pathways. The gene manipulation techniques developed and optimized on the INVSc1 platform laid a solid technical foundation for the subsequent seamless transition to the engineering modification of S. boulardii. And S. boulardii, as a widely used medicinal probiotic globally, has over several decades of extensive clinical safety data support, and its safety as a drug delivery carrier has been confirmed.

In summary, the current research strategy using INVSc1 is a reasonable and efficient path from "principle verification" to "product development". The successful concept verification will directly guide us to transplant the entire system to S. boulardii, which has excellent intestinal survival ability and proven safety, in the next step, ultimately achieving the clinical application goal of targeted drug delivery in the intestine.

Although Saccharomyces boulardii has a good overall safety profile in clinical applications, literature reports indicate rare but serious risks in specific high-risk populations. Key risk factors include ICU admission, total parenteral nutrition (TPN), central venous catheters, and immunosuppressive conditions. A systematic review of 117 cases of Saccharomyces boulardii fungemia between 2005 and 2022 revealed that 67.6% (n=73) occurred during probiotic administration, with an all-cause mortality of 36.1% (Vinayagamoorthy et al., 2023). Although the overall incidence of these adverse events is very low (representing 0.1-3.6% of all fungemia cases) (Riquelme et al., 2003), the consequences can be severe for immunocompromised patients.

Protection

We selected a yeast strain with four nutritional auxotrophies as the chassis for our engineering yeast. The yeast strains used as the chassis for the engineering yeast have defects in the autonomous synthesis ability of the four essential nutrients, namely leucine, tryptophan, uracil, and histidine. After the plasmid transformation operation was completed, the engineered yeast constructed in the laboratory still retained at least two synthetic defects (histidine and leucine). This characteristic makes the engineering yeast unable to autonomously synthesize the essential nutrients in the natural environment, and can only survive under the specific nutritional conditions controlled artificially in the laboratory, thereby effectively avoiding the risk of accidental leakage of the engineered yeast into the natural environment. It is worth noting that this nutritional deficiency design is only present in the laboratory model strain INVSc1 used in this study. It is an intentional setting for biosecurity considerations. However, in the Saccharomyces boulardii strain that will ultimately be used for practical applications, it inherently possesses complete metabolic capabilities and does not have such nutritional deficiencies. Furthermore, the strain in this laboratory does not possess the ability for conjugation transfer, nor does it produce spores with high environmental tolerance. This further eliminates the possibility of its spread in the environment through horizontal gene transfer or spore dissemination at the genetic level.

Countermeasure

This illustration comprehensively elaborates the complete molecular pathway for inducing programmed cell apoptosis through the optimized Tet-On system in engineered yeast. The core process begins with the diffusion of exogenous doxycycline (Dox) into the cell, which binds to the reverse-tetracycline-controlled trans-activating protein (rtTA3) in the cytoplasm. This fusion protein consists of a mutated TetR domain, a herpesvirus-derived VP16 strong transcriptional activation domain, and a nuclear localization signal (NLS); Dox binding induces a conformational change, exposing the DNA binding site. Subsequently, the activated rtTA3-Dox complex is guided by the NLS to actively transport into the nucleus and specifically recognizes and binds to the tetR operator sequence integrated on the chromosome, thereby strongly recruiting the transcription machinery to initiate the transcription of downstream apoptotic effector gene human Bax. The generated Bax mRNA is exported through the nuclear pore to the cytoplasm, and the translated Bax protein translocates to the outer mitochondrial membrane and undergoes oligomerization, forming a lipid channel, leading to mitochondrial outer membrane permeabilization (MOMP). This critical event triggers the release of mitochondrial intermembrane contents (such as cytochrome C) into the cytoplasm, ultimately irreversibly executing cell programmed death. This circuit achieves strict and precise temporal control of cell survival, and its "no-leakage, high-sensitivity" characteristic provides an efficient and safe "suicide switch" for gene therapy and synthetic biology applications based on yeast.

Operation and Control Safety

Foundation

All the laboratories that provided support for our iGEM project have successfully been incorporated into the school's strict and multi-dimensional safety supervision system, and have continuously passed various regular comprehensive reviews and irregular random spot checks organized by the system. This has provided a fundamental guarantee for the safe conduct of our experimental activities. These inspections deeply cover every key aspect of laboratory operation, including but not limited to: routine maintenance and standardized use of equipment, compliance layout of water and electricity lines and regular leak detection, effective configuration of fire-fighting equipment and absolute unobstructed emergency passages, as well as complete emergency showers and eyewash stations. All these rigorous measures jointly ensure that our hardware facilities and management norms fully comply with and even exceed all safety regulations, thus building an absolutely reliable, compliant and solid safety operation environment for the cutting-edge synthetic biology exploration.

Compliance

This laboratory fully implements the safety guidelines of iGEM in all its activities, strictly adhering to its safety regulations, and ensuring that all experimental procedures comply with international biosafety standards. At the operational level, all personnel strictly follow the standard biosafety procedures, including but not limited to wearing personal protective equipment as per regulations, using biosafety cabinets correctly, and strictly avoiding any violations. In terms of reagent management, we have established a strict classification storage system, storing hazardous reagents separately and equipping them with comprehensive emergency response plans. Additionally, laboratory waste is disposed of in accordance with iGEM safety requirements, completely eliminating the possibility of environmental pollution. Through systematic safety management measures, we continuously ensure the safety and compliance of the experimental process, providing a reliable guarantee for synthetic biology research.

Certification

This project has been officially approved by the college's biosafety committee through rigorous review, and has been awarded a dual-language (Chinese and English) version of the "Biosafety Commitment Letter". This document solemnly states: All experimental activities of this project do not involve the cultivation of pathogenic microorganisms or any experiments involving infectious substances. The project team and the college have both committed to strictly abiding by all biosafety regulations and operational norms promulgated by the state and the school, ensuring that the research work is carried out safely and in compliance.

Qualified

All the wet-lab members of our team have received systematic training in professional compulsory courses such as "Biochemical Experiments" and "Cell Biology Experiments". The course content comprehensively covers experimental operation norms and safety management knowledge, laying a solid practical foundation for the team. In addition, all members have successfully passed the 2025 laboratory safety access examination of Nankai University and obtained corresponding qualification certificates, achieving 100% qualification access. This indicates that not only do the team members have a systematic experimental training background, but they also all meet the safety job qualification requirements stipulated by the school. Throughout the entire experimental process, we have strictly adhered to all regulations and fully followed safety standards, effectively ensuring the smooth progress of the research work.

Management

This laboratory has always placed safety and regulations at the core of its daily management, and has established an efficient and rigorous regular operation mechanism. At the management level, experienced mentors who guide senior graduate students are responsible for regular supervision. They will regularly and promptly remind and train all members on key matters such as experimental operation norms, personal safety protection, and waste classification and disposal procedures; in team collaboration, members have also established a good atmosphere of mutual supervision and proactive reminders, ensuring that each step adheres to safety standards. In addition, the laboratory attaches great importance to the safety warning role of the environment. All key operation areas and equipment are marked with clear and explicit safety warnings and risk warning signs, forming a visual immediate reminder system. Thus, in terms of both institutional management and cultural atmosphere, it jointly ensures the high standardization and safety of the laboratory's daily operations, laying a solid foundation for the smooth conduct of all scientific research activities.

Control

This laboratory strictly adheres to the highest safety standards and takes meticulous measures to prevent the spread of harmful chemicals and biological materials such as yeast. All experimental waste, including discarded microbial cultures, chemicals, and consumables, are strictly categorized, inactivated, and centrally handled. They are managed by designated personnel and entrusted to qualified entities for standardized disposal, completely eliminating environmental pollution and biological safety risks, ensuring the safe and compliant operation of scientific research.

Reference

Details

Riquelme, A. J., Calvo, M. A., Guzmán, A. M., Depix, M. S., García, P., Pérez, C., Arrese, M., & Labarca, J. A. (2003). Saccharomyces cerevisiae fungemia after Saccharomyces boulardii treatment in immunocompromised patients. Journal of Clinical Gastroenterology, 36(1), 41–43. doi: 10.1097/00004836-200301000-00013

Vinayagamoorthy, K., Pentapati, K. C., & Prakash, H. (2023). Epidemiology of Saccharomyces fungemia: A systematic review. Medical Mycology, 61(2), Article myad014. doi: 10.1093/mmy/myad014