Introduction

Safety is of utmost importance for any laboratory work in the field of synthetic biology. Since our project relies on the use of genetically modified bacteria, both planning and execution of experiments was carried out with thorough attention to the necessary safety regulations and requirements.

EU law such as Directive 2009/41/EC, along with German laws including BioStoffV (Biological Agents Ordinance) or GenTG (Genetic Engineering Act) provide a robust framework for safe laboratory practices. Prior to commencing any laboratory work, microorganisms suitable for the planned experiments were selected to meet the project’s biological and chemical requirements while minimizing risk to potentially exposed individuals.

In line with these regulations, the microorganisms were contained and disposed of in accordance with the assessed safety level, taking aspects such as the organism type and specific genetic modifications applied in consideration. All procedures were carried out under strict documentation protocols. Each experiment was conducted in a BSL-2 (Biosafety Level) certified facility, with every experimenter having received appropriate training beforehand.

Meticulous planning combined with continuous supervision and conscientious laboratory practices ensured that all work was conducted in accordance with modern standards of safe biological experimentation. The following sections provide a more detailed elaboration of our safety concept.

Biosafety and Containment

Organisms and Lab Containment

All laboratory work was performed using BSL1 organisms; however, experiments were conducted in a BSL-2 laboratory to ensure additional safety. Core chassis strains comprised Escherichia coli K12 derivatives and Bacillus subtilis W168 (trpC2), which were employed for gene cloning, expression, and secretion experiments. Additionally, Solibacillus silvestris CGN12 was used for benchtop microbially induced calcite precipitation (MICP) assays, and Agrobacterium sp. ATCC 31749 served as a reference for curdlan biosynthesis studies. Each organism was handled within sealed systems with appropriate containment measures, without introduction of mobile genetic elements and with exclusion of traits related to pathogenicity or environmental persistence. In our assessment, given the exclusive use of BSL1 organisms under closed system containment and validated inactivation procedures, the potential environmental risk of the work was assessed as minimal.

Human Health Risk

Considering the non-pathogenic nature of all strains used and the absence of mammalian targeted toxins or virulence factors in our constructs, we assessed the risk to human health as minimal. Established laboratory practices and containment procedures further mitigated potential exposures for personnel.

Environmental Safeguards

All procedures were conducted in controlled laboratory environments with no release of organisms or genetic material into the environment. Constructs excluded elements that could facilitate horizontal gene transfer or confer enhanced environmental fitness. Based on our project’s risk assessment, the potential environmental impact was assessed as minimal.

For a safe implementation in actual construction, a mazEF-type kill-switch could be built into the bacteria to prevent unwanted spread of strains in the environment (Kill-Switch | SublimeStone). The kill-switch is a toxin-antitoxin system comprised of two genes: mazE (the antitoxin) and mazF (the toxin). Under normal conditions, MazE binds MazF, thereby neutralizing the toxin. However, under stress—such as oxidative stress or antibiotic exposure—the antitoxin is degraded by proteases such as ClpPA or Lon. In the absence of the antitoxin, MazF proceeds to cleave single-stranded RNA, which leads to inhibition of bulk protein synthesis and consequent growth arrest. Additionally, bacteria can be modified to synthesize cell-death proteins that are encoded by mRNA that is not cleaved by MazF, resulting in cell death. Implementation of this system in bacteria used in this project would create an additional level of safety.

mazEF kill-switch mechanism (Engelberg-Kulka et al., (2005)).

Lab Safety and Operations

Training and Access

Those team members who performed wet lab work had completed the required documented safety training under the German Biostoffverordnung and the Gentechnikgesetz before beginning their laboratory tasks. Access to genetically modified organisms, hazardous chemicals, and specialized equipment was restricted to trained and authorized personnel. Personal protective equipment, including lab coats, gloves, and eye protection, was required throughout all experimental procedures.

Chemicals and Waste

Biological and chemical materials were handled in accordance with the EU CLP Regulation (1272/2008). Chemicals that posed a health hazard were handled strictly within a fume hood and disposed in accordance with regulations, depending on the type of the substance. Where feasible, lower risk DNA stains such as GelRed or SYBR Safe were used. Antibiotics and chemical inducers like bacitracin were applied at the lowest concentration necessary, followed by complete inactivation and proper disposal. Laboratory waste—including media, disposables, and GMO containing materials—was autoclaved prior to disposal according to institutional procedures. Surfaces and equipment were promptly disinfected in case of contamination or spills, with established protocols ensuring effective emergency response and documentation.

Compliance and Records

Documentation and Compliance

Complete and accurate records were maintained for all strains, plasmids, and genetic modifications, including sequence verification and storage information, to ensure traceability. Compliance with institutional biosafety requirements and applicable legal provisions was monitored continuously. Any protocol changes that could alter the risk profile underwent prior assessment and approval.