Introduction

Synthetic biology, not only in the iGEM competition but also in industry revolution, is an interdisciplinary field dedicated to designing and constructing novel biological systems beyond those found in nature. While it offers tremendous promise, it also carries significant risks to human health and the environment which must be carefully managed. Biosafety, often described as a “Sword of Damocles” hanging over the world, demands constant vigilance^[1]. We have developed our project around Grape Yeast (detailed on our Design page), and integrated safety considerations into every stage to help shape a more responsible future for synthetic biology.

Since our experimental design and application scenarios take place entirely within the laboratory, we emphasize on laboratory safety. Beyond standard laboratory safety measures, we conduct project-specific biosafety assessments and implement tailored risk-mitigation strategies.

Laboratory Safety

Laboratory safety is an essential part of every iGEM project, which underscores its critical importance.

Through our literature research, we became convinced that laboratory safety is a field that deserves far more systematic attention. Every year, accidents occur in laboratories worldwide — some causing minor injuries, others posing serious risks to life. Yet, we were surprised to see that while many iGEM teams touch on this topic, they often limit their discussion to listing safety trainings or regulations, without providing a structured or holistic perspective on laboratory safety.

This year, we set out to go beyond this. Our aim is to develop practical models and solutions that can improve laboratory safety and serve as a lasting reference for future teams working in this important area.

To achieve this, we applied the 24Model, a popular systemic accident model in China^[2], systematically identifying potential risks in our Grape Yeast project and addressing them through multi-level oversight and layered management. To extend our impact beyond our own lab, we also conducted a university-wide survey on laboratory safety awareness and developed an SOP-based solution package that can be adopted by others.

In the 24Model^[2:1] framework, safety evaluation is conducted as outlined in the table below.

Table 1. 24Model's Laboratory Safety Evaluation Dimensions

Dimension Level	Main Categories	Key Evaluation Areas	Description
Individual Level	One-time Behaviors & Conditions	- Unsafe Human Behaviors - Unsafe Physical Conditions	Direct causes of accidents that are immediate unsafe actions or hazardous conditions triggering incidents.
Individual Level	Habitual Behaviors	- Deficiencies in Safety Knowledge - Weak Safety Awareness - Unsafe Laboratory Habits - Unfavorable Psychological/Physiological States	Indirect causes due to long-term unsafe habits or persistent weaknesses that under specific circumstances lead to unsafe acts or conditions.
Organizational Level	Safety Management System	- Organizational Structure Gaps - Responsibility Allocation Gaps - Procedural/Documentation Deficiencies - Resource Management Deficiencies - Training Deficiencies	Root causes related to systemic weaknesses in management, regulations, procedures, and resources.
Organizational Level	Safety Culture	- Value of Safety - Belief that Accidents Are Preventable - Safety as Good Practice - Leadership & Oversight Responsibility - Personnel Safety Performance	Fundamental root causes shaped by safety culture, impacting how safety is prioritized and integrated across the organization.

As part of this effort, we compiled a Laboratory Safety Analysis Report, available as a PDF file. We hope it will offer future iGEM teams and laboratory practitioners a systematic perspective on laboratory safety, as well as fresh insights for building safer research environments.

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Following the systematic risk assessment, we recognized that structured training and tiered management are essential for creating a robust safety culture. By ensuring that every member is both well-prepared and forward-thinking, we can sustain a culture of safety.

1.1 A Tiered Training Program

University-Level Training: Before entering the lab, every member completes University's mandatory laboratory safety course and examination. This program covers:

• Proper use of personal protective equipment (PPE)
• Safe operation of common laboratory instruments
• Handling of chemicals, biological materials, and waste
• Emergency response to fire, electrical hazards, and spills

Passing the course ensures all participants have the baseline skills and awareness to respond effectively to safety issues.

School-Level Training: Students working on wet-lab experiments must pass the School of Life Sciences' specialized safety examination before gaining lab access. Training topics include:

• Prohibited laboratory practices
• Safe storage and handling of hazardous chemicals
• Waste management and daily lab operations
• Equipment guidelines and fire safety measures

The school conducts weekly safety inspections, to reinforce compliance and create a culture of accountability.

Laboratory-Level Training: Upon joining the lab, members receive hands-on safety orientation from instructors. Safety measures include:

• Posted reminders at key equipments such as ultra-low temperature freezers, biosafety cabinets, and centrifuges
• Mandatory use of lab coats, blue nitrile gloves, and clean benches
• Post-experiment cleaning and proper waste disposal
• Removing PPE and washing hands before leaving the experimental areas
• Regular UV-light disinfection of specific lab spaces

New members also need to complete a Good Lab Practice Quiz, helping them master essential skills while protecting both themselves and the equipments.

1.2 Safety Resources and Documentation

University-Level Resources: Fudan maintains a Laboratory Safety Education and Management Platform featuring:

• A comprehensive safety manual and SOPs for key equipment
• Laboratory policies and regulations
• Online training modules and assessments

Laboratory-Level Resources: Our lab supplements university resources with its own Emergency Response and Safety Guidelines, providing detailed instructions for preventing and managing incidents:

• Beginner-friendly introductions to molecular biology
• Step-by-step experimental protocols
• Microscopy imaging tutorials
• Laboratory safety manuals

Building a Culture of Safety

By layering University, School, and Laboratory safety programs, we've created a comprehensive, continuous, and proactive safety framework. This ensures all members receive the training and resources they need to work safely while advancing responsible and sustainable science.

Project-Specific Biosafety Assessment & Measures

Oversight of emerging technologies is constantly evolving, as the risks they pose are often not yet fully known or analyzed. For biosafety risk management, this means mitigation strategies must consider both the proven risks and the potential consequences that experiments may — or may not — produce.

While Saccharomyces cerevisiae is a well-established chassis in synthetic biology, multicellular yeast is being introduced as a chassis for the very first time to iGEM. Because of this, no team has yet explored or prepared for its associated risks. As our chassis organism, Grape Yeast presents uncharted challenges, and we seek to take the lead in assessing these risks and providing responsible solutions.

Our approach is structured around the following key areas:

• Designing multilayered genetic circuits
• Ensuring the biosafety of the apoptotic element BAX in yeast
• Reducing reliance on hazardous reagents

2.1 Multilayered Genetic Circuits

In synthetic biology, biocontainment strategies are generally built around three approaches: auxotrophy, essential gene regulation, and toxin expression.

While these approaches can be highly effective, each comes with limitations: Auxotrophic strains may regain growth ability through cross-feeding of metabolites or the presence of required molecules in the environment. Essential genes can sometimes exhibit leaky expression, allowing unintended survival. Meanwhile, mutations may disable toxin systems. Redundancy can lower escape risks, but often at the cost of reduced fitness, giving escape mutants a growth advantage.

To address these challenges, design a robust genetic safeguards should meet three key criteria: low escape frequency, robustness, and modularity.

• Low escape frequency ensures that mutants cannot thrive outside of controlled media and prevents their spread in natural environments.
• Robustness maintains wild-type fitness levels while ensuring containment across different growth conditions, even in nutrient-rich or complex environments where other organisms may supply missing metabolites.
• Modularity allows multiple safeguard strategies to be layered within a single strain or transferred across different hosts, making safeguards more versatile and portable.

Inspried by the Gallagher et al's circuit^[3], which integrates three core modules: a riboregulatory system, a biotin auxotrophy strategy, and a nuclease-based kill switch.

• Riboregulatory module: The pLtetO promoter is repressed by TetR and activated by aTc to express trans-activating RNA (taRNA). Meanwhile, the pLlacO promoter is repressed by LacI and activated by IPTG to drive cis-repressing RNA (crRNA) along with an essential gene. The taRNA and crRNA interact through a looped intermediate structure that exposes the crRNA's ribosome binding site (RBS), enabling expression of the downstream essential gene.
• Biotin auxotrophy module: Normally, bioA produces the precursor of biotin's heterocyclic ring, while bioB catalyzes the final ring closure to generate active biotin. Disruption of both genes creates an auxotrophic strain that requires exogenous biotin for growth.
• Nuclease kill-switch module: Constitutive expression of EcoRI nuclease kills cells unless protected by EcoRI methyltransferase, which is itself placed under aTc regulation.

Together, in their original design include these modules ensure that E. coli can only survive when all three inputs — aTc, IPTG, and exogenous biotin — are present simultaneously. This layered safeguard prevents accidental survival caused by the leakage of any single module.

The circuit^[3:1] was originally designed in E. coli, since our chassis is Saccharomyces cerevisiae, we adapted the system to function in yeast. The table below summarizes the modifications across the three modules:

Table 2. Proposed Modifications to Multilayered Safeguards

Module	Original E. coli Design	Adapted S. cerevisiae Design	Key Modifications
Module I: Riboregulatory Circuit	- Controlled by pLtetO + TetR and pLlacO + LacI - crRNA and taRNA interact through a loop structure to regulate essential gene expression	- Replaced with a CRISPRi-based Rz–gRNA–Rz construct, where ribozymes self-cleave to process functional gRNA - The 3′ HDV ribozyme was substituted with an Aptazyme, which responds to small molecules. Binding prevents cleavage, leading to gRNA inactivation and de-repression of the target	- Transitioned from RNA–RNA interaction to CRISPRi precision control - Introduced a small-molecule-responsive Aptazyme to add a conditional regulatory layer
Module II: Auxotrophy	- bioAB deleted and replaced with bla; - loss of bioAB prevents biotin synthesis, making growth dependent on exogenous biotin	Switched to URA3 auxotrophy, restricting yeast growth to media supplemented with uracil	Shifted from biotin dependency in E. coli to uracil dependency in yeast, aligning with yeast's common auxotrophic markers
Module III: Kill Switch	- Based on EcoRI endonuclease, with EcoRI methyltransferase under aTc control - Without methylation, EcoRI cuts DNA, causing cell death	- Implemented the CamOff-RelE system: RelE, an mRNA endonuclease, cleaves translating mRNA to halt protein synthesis, leading to cell death - Camphor serves as the external inducer, controlling the CamOff switch	- Replaced a DNA-targeting enzyme with an mRNA-targeting toxin (RelE) - Applied the CamOff inducible switch for precise, small-molecule-regulated control

Adapting from E. coli to Saccharomyces cerevisiae

Module I: Redesign of the Riboregulatory Circuit

To replace the crRNA/taRNA system, we turned to CRISPRi, which enables precise control of gene expression using dCas9 and gRNA. Importantly, CRISPRi only activates in response to specific triggers—exactly matching the conditional control we aimed for.^[4]

The design links ribozymes to both ends of the gRNA (Rz–gRNA–Rz). These ribozymes self-cleave after transcription, trimming the gRNA into a clean, functional form. Any disruption to this process prevents proper maturation, effectively “switching off” the gRNA.

Building on this framework, the literature substituted the 3′ HDV ribozyme with an Aptazyme—a hybrid RNA molecule that combines a ribozyme with a small-molecule-binding aptamer. Downstream, the retained the poly(A) tail and inserted an antisense RNA (asRNA) sequence.

• Without small molecules: The Aptazyme's ribozyme remains active and cleaves as normal. This removes the poly(A) tail and asRNA, producing functional gRNA. The system stays in a repressed state (GFP OFF).
• With small molecules theophylline: Binding of the ligand alters the Aptazyme's structure, shutting down ribozyme activity. Without cleavage, both the poly(A) tail and asRNA remain. The poly(A) tail directs the transcript for degradation, while the asRNA pairs with the gRNA backbone, blocking dCas9 binding. Together, these mechanisms render the gRNA inactive, dCas9 is no longer guided to its target, repression is lifted, and the system flips to a de-repressed state (GFP ON).

Module II: Nuclease-Based Kill Switch

To restrict growth of our chassis, we replaced the traditional biotin auxotrophy with a URA3-based marker. This modification ensures that Grape Yeast can only survive on media supplemented with uracil, effectively confining its growth to tightly controlled laboratory conditions.

Module III: Nuclease-Based Kill Switch

According to the literature, the CamOff-RelE system functions as a highly tunable “suicide switch.”^[5] In this design, camphor acts as the central regulatory molecule. Camphor is an aromatic terpenone that occurs naturally in the bark of camphor trees, but is now easily synthesized and inexpensive. The CamOff system harnesses the natural transcriptional regulation of the Cam repressor from Pseudomonas putida. The toxin component, RelE, is a Type II ribonuclease that cleaves actively translating mRNA. When expressed in Saccharomyces cerevisiae, RelE has been demonstrated to be lethal. Thus, by simply adding or withholding camphor, the survival of the engineered system can be tightly controlled.

Building on Fudan iGEM 2020's success, we are confident to adapt Multilayered Safeguards to our yeast. However, due to the limit of time and we could not implanted it before 2025 Oct.

Towards Real-World Applications

After completing our design, we consulted Professor Tang, a leading expert in biosafety and ethics in synthetic biology (details on our integrated human practices page). We asked for her perspective on our circuit design — whether it meets industrial needs, and what challenges it might face in moving from laboratory proof-of-concept to real-world application.

Our discussion with Professor Tang was eye-opening. We realized that genetic safeguards exist within a complex web of stakeholders, each with their own priorities. For synthetic biologists, safeguards are primarily proof-of-concept tools designed to restrict the growth of engineered strains or prevent horizontal gene transfer. Regulators and policymakers often treat them as data sources for risk assessment and policy-making, while actual regulatory adoption remains distant and highly context-dependent. From an industry standpoint, safeguards are often seen as unnecessary in physically contained applications already covered by infrastructure and regulation; meanwhile, novel applications are still considered too theoretical to fit within existing risk assessment frameworks. Civil society is barely represented in these discussions, yet both developers and end users remain cautious about how the public might perceive new safeguard technologies.

This divergence in perspectives highlights a deeper issue: the lack of collective reflection on the actual needs and expectations surrounding safeguards. As academic researchers, we cannot rely on an idealistic outlook. Instead, we must actively engage stakeholders early on in conversations about the relevance and practicality of safeguards. Frameworks like Responsible Research and Innovation can help ensure that research remains aligned with broader socio-economic realities, while also guiding the balance of social and economic forces needed to bring innovation into practice.

Figure 3. Discussion with Professor Tang

After the discussion, we began to ask ourselves: what additional challenges must be addressed if we want to bring this concept into real-world applications?

Current literature highlights a significant gap — although many biosafety safeguard systems have been proposed in synthetic biology, there is still no standardized way to test whether these systems are truly reliable. The most pressing question remains: how can we measure and evaluate their long-term effectiveness?

One challenge for the field is our limited ability to monitor and control synthetic organisms once they are released. Equally important is the need to understand how these organisms behave in real ecosystems. Once introduced, they may compete with native species for resources, spread disease, act as predators, or themselves be preyed upon. Anticipating and evaluating such interactions is critical.

Another concern is horizontal gene transfer — the unintended spread of engineered genes into natural organisms. Current strategies to prevent this remain scarce, with only a recently developed approach based on genetic code similarity offering a potential way forward.^[5:1]

For our Grape Yeast, we identified two specific countermeasures:

Genetic Barcode

To ensure traceability, we hope to introduce unique, irreversible DNA barcodes into the genome of Saccharomyces cerevisiae. These short (~20 bp) sequences are easily identified by PCR or high-throughput sequencing, allowing us to rapidly detect and track engineered strains if they escape or appear in environmental samples. By linking the barcode directly to engineered loci or placing it at stable chromosomal positions, the barcode serves as a permanent “identity tag” for the strain.^[6]

During the assembly of Level 0 plasmids into higher level constructs, we used Golden Gate assembly connectors. Each connector carries 4-bp sticky ends generated by type II restriction enzymes, which can also function as programmable barcodes when incorporated into the genome.

Genomic Integration over Plasmids

To minimize biosafety risks, we chose chromosomal integration as the primary method for implementing our engineered parts, rather than plasmids.

Plasmids are among the main carriers of horizontal gene transfer (HGT) in microbial communities. Although yeast has relatively few self-transmissible plasmids, plasmids can still vary in copy number and stability, and under certain conditions exchange DNA with difference species. This poses ecological risks if engineered strains were released. By integrating engineered parts into the genome as chromosomal copies, we improve stability and reduce the likelihood of horizontal transmission or long-term persistence in the environment. Comparative studies also support that genome integration offers more reliable expression and control for complex, multigene pathways.^[7]

Thus, we adpot the designs in the Yeast Toolkit for Modular, Multipart Assembly (YTK)^[8] and the Multiplex Yeast Toolkit (MYT)^[9], we integrated promoters, resistance markers, terminators, and other functional elements directly into the genome. This approach decreases plasmid loss and enhances long-term stability.

2.2 Ensuring the Safety of BAX-Mediated Apoptosis in Yeast

The apoptosis gene BAX, which we introduced to multicellular yeast for controlling its cluster size. To address concerns about potential unintended side effects, we placed BAX under the control of an inducible Tet2 promoter^[9:1]. This promoter has a low level of basal (leaky) expression when no inducer is present, which helps minimize risks by preventing unintended activation.

2.3 Minimizing the Use of Hazardous Reagents

To protect researchers during laboratory work, we avoided the use of hazardous reagents such as phenol-chloroform when purifying yeast genomic DNA for PCR purpose. These chemicals are toxic and pose serious health risks, including carcinogenicity and central nervous system depression through inhalation. By using a protoocl without phenol-chloroform, we reduce potential harm to experimenters.

Align well with iGEM's Safety Recommendations

• We have applied a systems-thinking approach to lab safety
• We ensured all members completed lab safety training before any wet-lab experiment
• We propose a multilayered safeguards for yeasts (due to the limit of time, we have not implanted it in our current version)
• We used homologous recombination to stably integrate DNA fragment into desinated loci of the yeast genome, and all mistargeted yeast strains were discarded, reducing risks of genetic leakage and horizontal transfer

Reference

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1. Yu, Y., Ding, J., Zhou, Y., Xiao, H., & Wu, G. (2022). Biosafety chemistry and biosafety materials: A new perspective to solve biosafety problems. Biosafety and health, 4(1), 15–22. DOI: 10.1016/j.bsheal.2022.01.001 ↩︎

2. Fu, G., Xie, X., Jia, Q., Li, Z., Chen, P., Ge, Y. (2020). The development history of accident causation models in the past 100 years: 24Model, a more modern accident causation model. Process Saf Environ Prot, 134, 47–82. DOI: 10.1016/j.psep.2019.11.027 ↩︎ ↩︎

3. Gallagher, R. R., Patel, J. R., Interiano, A. L., Rovner, A. J., & Isaacs, F. J. (2015). Multilayered genetic safeguards limit growth of microorganisms to defined environments. Nucleic acids research, 43(3), 1945–1954. DOI: 10.1093/nar/gku1378 ↩︎ ↩︎

4. Xi, C., Chiu, S., Voje, W. E., Carothers, J. M., & Moon, T. S. (2025). Conditional guide RNA deactivation by mRNA and small molecule triggers in Saccharomyces cerevisiae. New biotechnology, 89, 105–118. DOI: 10.1016/j.nbt.2025.07.004 ↩︎

5. Asin-Garcia, E., Kallergi, A., Landeweerd, L., & Martins Dos Santos, V. A. P. (2020). Genetic Safeguards for Safety-by-design: So Close Yet So Far. Trends in biotechnology, 38(12), 1308–1312. DOI: 10.1016/j.tibtech.2020.04.005 10.1016/j.tibtech.2020.04.005 ↩︎ ↩︎

6. Pierce, S. E., Davis, R. W., Nislow, C., & Giaever, G. (2007). Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures. Nature protocols, 2(11), 2958–2974. DOI: 10.1038/nprot.2007.427 ↩︎

7. Harrison, E., & Brockhurst, M. A. (2012). Plasmid-mediated horizontal gene transfer is a coevolutionary process. Trends in microbiology, 20(6), 262–267. DOI: 10.1016/j.tim.2012.04.003 ↩︎

8. Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS synthetic biology, 4(9), 975–986. DOI: 10.1021/sb500366v ↩︎

9. Shaw, W. M., Khalil, A. S., Ellis, T. (2023). A Multiplex MoClo Toolkit for Extensive and Flexible Engineering of Saccharomyces cerevisiae. ACS Synth Biol. 12(11), 3393-3405. DOI: 10.1021/acssynbio.3c00423 ↩︎ ↩︎