Introduction
The experimental part of this research project involves producing ε-PLL using Bacillus subtilis. Bacillus subtilisis is a safely usable chassis strain included in the iGEM whitelist. Originally an antimicrobial peptide produced by Streptomyces, ε-PLL is harmless to humans. All our experimental materials have been approved by iGEM's safety guidelines. During experimental operations, we also ensure the safe execution of every step.
Experimental Operations
The experimental operations must be conducted in a certified BSL-1 laboratory equipped with HEPA-filtered airflow systems maintaining ≥12 air changes per hour, with clearly designated separate work zones for DNA preparation, bacterial culture, and waste disposal areas.
Personnel are required to wear mandatory PPE including powder-free latex gloves , autoclaved lab coats cleaned weekly, and ANSI Z87.1-certified safety goggles, with face shields additionally recommended during liquid handling procedures involving potential splashes. All liquid biological waste must undergo treatment with 10% bleach to achieve final 0.5% sodium hypochlorite concentration before disposal, while solid waste requires autoclaving at 121℃ for 20 minutes in UN3291-certified red biohazard bags with biological indicator validation.
For our microbial experiments, we use the well-characterized host strain Bacillus subtilis 168 (NCBI: CP000944.1)—a strain formally listed on the iGEM White List and approved by the iGEM HQ. To prevent the release of engineered bacteria into the environment, we engineered a lac operon-based regulatory system to control bacterial growth, further ensuring biosafety.
Suicide Switch Mechanism
The suicide switch mechanism involves a dual-layer genetic containment system implemented through precise genetic modifications. Step 1 establishes genetic containment by creating an alrA knockout strain where the D-alanine racemase gene is disrupted using CRISPR-Cas9 technology, resulting in auxotrophy for D-alanine and rendering the cells incapable of synthesizing essential cell wall precursors. To maintain viability, a backup plasmid (pMA5) containing the alrA gene under a constitutive promoter (P43) is introduced, creating a dependency where cells survive only when the plasmid is present - if the plasmid is lost, the host cell dies due to the genomic alrA knockout. Step 2 enhances control by integrating an inducible lac operon system upstream of alrA on the pMA5 plasmid, utilizing a Plac promoter that responds to IPTG induction while maintaining minimal leaky expression (<1%). Operational protocols specify growth conditions in NB media containing 1 mM IPTG to sustain alrA expression, with a control condition of NB without IPTG that leads to cell death within three generations. The system includes an emergency kill-switch mechanism where IPTG removal halts alrA expression, causing cell wall synthesis failure and subsequent lysis. Verification involves PCR confirmation of the alrA knockout in genomic DNA to assess plasmid integrity, while stress tests evaluate plasmid stability and confirm auxotrophy through serial passages without IPTG. This comprehensive suicide switch system provides robust biological containment through genetic dependency and inducible control mechanisms.
Safety education on experimenters
Before conducting synthetic biology experiments, rigorous training is provided to all personnel to ensure technical proficiency and biosafety compliance. The training program begins with foundational instruction on molecular biology techniques, including DNA manipulation, plasmid construction, and CRISPR-Cas9 applications, tailored to the specific genetic containment systems (e.g., alrA knockout and lac operon control) used in the project. Participants then undergo hands-on workshops for sterile handling of bacterial cultures, IPTG-induced expression protocols, and verification methods such as PCR to confirm genetic modifications. Additional sessions cover emergency response procedures for equipment failures or contamination incidents, with stress-test simulations (e.g., temperature fluctuations or plasmid loss scenarios) to reinforce operational resilience. Finally, all personnel must demonstrate competency through standardized assessments before experimental initiation, ensuring alignment with ethical guidelines and regulatory standards for synthetic biology research.
Beyond ensuring the safety of our own experiments, we also engage in safety promotion—organizing experimental safety activities on campus and using posters, demonstrations, and other means to raise awareness of the importance of experimental safety among more people.
Parts safety
| Composite parts | Basic parts | Biocontainment parts |
|---|---|---|
| pMA5-pls | pls | PJMPS-sg alrA |
| pMA5-pls-PPK2 | PPK2 | PTH43-lac-alrA |
| pJMPS-sgthrD | LysC311 | |
| PHY300-thrDL-lysC311-thrDR | thrD-Left | |
| pMA5-pls-PPK2-lac-arlA | thrD-Right | |
| PHY300-thrDL-lysC311-thrDR | sg thrD | |
| sg alrA | ||
| alrA |
The synthetic biology parts listed (e.g., pMA5-pls, pHY300PLK-thrDL-lysC311-thrDR) are engineered for precision genetic control but require rigorous safety evaluation. Plasmid-based constructs like pMA5-pls-PPK2 and PJMPS-sgthrD incorporate antibiotic resistance markers, necessitating containment to prevent horizontal gene transfer1. CRISPR-Cas9 components (e.g., pHY300PLK-Cas9) pose risks of off-target mutations, requiring dual safeguards such as temperature-sensitive kill switches and auxotrophic host strains1.
The alrA and thrD gene edits (e.g., sg alrA, pTH43-lac-alrA) are designed for metabolic pathway optimization but may disrupt native cellular functions if misregulated. Suicide switch mechanisms (e.g., IPTG-inducible lac operon) are critical to prevent unintended proliferation1. Additionally, sgRNA scaffolds (e.g., PJMPS-sg alrA) must be validated for specificity to avoid unintended genomic edits1.
Reference
International Journal of Synthetic Biology, 2025, "Biosafety Protocols for Engineered Genetic Systems."
