Safety & Security

Before initiating any experimental procedures, we conducted a thorough hazard assessment (Hazard Assessment and Containment Implementation – HACI) to identify all possible risks associated with bacterial handling, enzyme use, and cloning procedures.

Lab work was performed either:

• inside a laminar flow hood to prevent aerosol contamination,
• or on a designated “bacteria-only bench”, specifically organized for GMO handling.

At the bacteria bench, we followed strict sterile practices:

• A Bunsen like burner was kept continuously lit to maintain an upward air current and reduce the risk of contamination.
• All manipulations of E. coli cultures (e.g., streaking, pipetting, minipreps) were performed close to the flame.



• Waste containers were clearly labeled and separated specifically for bacterial materials, ensuring traceability and proper disposal.

These practices ensured a clean working environment, minimized cross-contamination, and helped contain genetically modified organisms effectively, even in open-bench conditions.

In Chitinator et al., we used antibiotics such as kanamycin and chloramphenicol as selection markers during bacterial culture and protein expression. Given their biological activity and the fact that they are coupled with GMO use, we treated all antibiotic containing waste with extra care.

We established the following waste management practices:

• Dedicated biohazard bins were placed in every bench where bacterial cultures were used.
• Solid waste (e.g., pipette tips, gloves, agar plates) contaminated with E. coli or antibiotics was collected in labeled biohazard containers and autoclaved before final disposal.
• Liquid cultures were collected in separate waste flasks, autoclaved, and only then discarded.
• Media and reagents with antibiotics were never disposed of in sinks, to prevent environmental contamination.
• Antibiotic stocks were stored safely, clearly labeled, and handled only by trained members.

We made sure to never discard untreated genetically modified material or antibiotics in the general waste stream. By following institutional and national biosafety guidelines, we minimized the environmental footprint of our lab work.

All team members working in the lab completed mandatory safety training prior to beginning any experimental work. This included:

• Proper use of personal protective equipment (PPE) such as lab coats, gloves, and face masks.
• Safe operation of key equipment including centrifuges, incubators, gel electrophoresis units, and sonicators.
• Specific protocols for handling genetically modified organisms (E. coli), antibiotics, and hazardous reagents.
• Emergency response procedures (fire extinguisher use, eye-wash station, chemical spill handling).

Standard Operating Procedures (SOPs) for each major experimental protocol were printed and displayed in visible locations across the lab. Additionally, new members were always supervised by trained individuals until they were fully confident to work independently.

Our training program ensured that everyone understood not just how to perform experiments, but how to do so responsibly and safely.

All wet-lab work for Chitinator et al. was carried out in a certified Biosafety Level 1 (BSL-1) laboratory. The only organisms used were Escherichia coli strains such as BL21 and Rosetta, all of which are non-pathogenic and widely accepted as safe for educational and research use.

To ensure full compliance with national and institutional biosafety regulations:

• All genetically modified organisms were stored in designated GMO refrigerators and freezers, clearly labeled and access-controlled.
• No GMOs were released into the environment. All cultures were inactivated before disposal via autoclaving or disinfectants.
• Every transformation, plasmid, and bacterial stock was logged in a shared lab book, indicating the date, construct, responsible team member, and antibiotic used.
• Transport of samples between labs was done using sealed containers, following our university’s safety policy.

Our adherence to BSL-1 protocols ensured that the project was not only innovative but also safe and ethically responsible.

For the purposes of enzyme expression in Chitinator et al., we selected non-pathogenic Escherichia coli strains, specifically BL21(DE3) and Rosetta(DE3). These strains are both classified as Biosafety Level 1 (BSL-1) and are widely used in synthetic biology and protein production due to their safety profile and high expression efficiency.

• E. coli BL21(DE3) is optimized for high-level expression of recombinant proteins and lacks the proteases Lon and OmpT, which makes it ideal for stable protein production.
• Rosetta(DE3) is a derivative strain that provides enhanced expression of eukaryotic proteins by supplying tRNAs for rare codons, making it particularly useful for expressing fusion enzymes with complex structures.

These strains were chosen not only for their technical advantages but also for their well-documented biosafety characteristics. Both are considered non-toxic, non-invasive, and environmentally safe under controlled laboratory use.

Importantly, all work involving these strains was performed exclusively within a certified BSL-1 facility, and bacterial cultures were contained entirely within a closed bioreactor system, with no release of live GMOs at any stage of the process. This choice reflects our team’s commitment to both experimental efficiency and biological responsibility.

In addition to our experimental work in E. coli, we also propose the potential use of Bacillus subtilis strain 168 as a host organism for future implementation of our system, especially in contexts such as soil treatment or large-scale bioreactor fermentation.

B. subtilis 168 is a model Gram-positive bacterium with a long history of safe use in research and industrial biotechnology. It is classified as a Biosafety Level 1 (BSL-1) organism and is Generally Recognized As Safe (GRAS) by international standards. It does not produce toxins or pose risks to human health or the environment.

By proposing B. subtilis 168 for the implementation phase, we aimed to ensure that every stage of our system , from design to deployment , aligns with biosafety and bioethics principles, making Chitinator et al. a safe and scalable solution for real-world applications.

For the first experimental phase of Chitinator et al., we selected the pET-28a(+) plasmid as our expression vector for use in E. coli BL21(DE3) and Rosetta(DE3). This plasmid is well-characterized, widely used in molecular biology, and contains features ideal for high-yield recombinant protein production, including:

• a T7 promoter for strong inducible expression,
• a Kanamycin resistance gene for selection,
• and a multiple cloning site compatible with our fusion chitinase gene construct.

In this phase, the use of antibiotics was confined strictly to laboratory settings under certified BSL-1 conditions, and all antibiotic-containing cultures were properly decontaminated prior to disposal.

For the implementation phase, where enzyme production would take place in a closed bioreactor, our aim is to shift to a plasmid system that is free of antibiotic resistance markers. This is particularly important in applications involving environmental interfaces, such as compost enrichment or agricultural soil use, where the use of antibiotic resistance genes is not appropriate.

In this context, we have considered Bacillus subtilis 168 as a suitable host for implementation due to its excellent secretion capabilities and BSL-1 classification. For experimental testing in B. subtilis, we selected the pHT43 plasmid, which is compatible with this chassis and enables secretion of recombinant proteins via the amyQ signal peptide.

However, we recognize that pHT43 contains a chloramphenicol resistance gene, and therefore is not suitable for field use. Our long-term goal is to transition to a plasmid system compatible with B. subtilis that does not rely on antibiotic-based selection, possibly through auxotrophic complementation, toxin-antitoxin systems, or inducible chromosomal integration strategies.

This dual-plasmid strategy, one for testing, one for implementation, reflects our commitment to safe, modular, and responsible synthetic biology, allowing us to optimize both the experimental workflow and future scalability of our system.

Our project centers around the controlled production of chitin-degrading enzymes within a closed bioreactor system using genetically modified E. coli. At no point do the bacteria exit the reactor or come into contact with the environment. The final product is a purified enzymatic extract, completely free of living GMOs.

This extract can be used in two main application areas:

1. As a compost enhancer, enriching organic waste with nutrients derived from chitin degradation (e.g., N-acetylglucosamine, carbon, nitrogen) and accelerating microbial activity in the composting process.
2. As a nutrient base for future bacterial consortia engineered for advanced biodegradation tasks; for example, bacteria expressing PETases or other enzymes that break down microplastics. By using the chitin-derived nutrients as a cultivation substrate, our system could support the growth of such strains in a safe and controlled way.

In both applications, the final material is safe, non-toxic, and entirely free of live genetically modified organisms. We strongly support the principle of zero environmental GMO release, designing all parts of our system to meet the highest standards of biosafety and end-user security.

From the beginning of our project, we considered not only the technical feasibility but also the ecological and social responsibility of our proposed solution. Chitin is a biodegradable polymer found in natural biomass, and its breakdown by chitinases releases nutrients that are beneficial to soil ecosystems.

To prevent any unintentional environmental release, all E. coli cultures used during our experiments were completely inactivated through autoclaving or chemical disinfection prior to disposal. All laboratory activities took place exclusively in a certified BSL-1 facility, with no contact between GMOs and the natural environment.

Importantly, our enzyme production system is designed to operate entirely within a closed bioreactor, ensuring that no genetically modified organism is ever released into the field. The resulting enzymatic extract, free of living cells, is intended for applications such as:

• enhancing compost with bioavailable nutrients, or
• serving as a substrate for other engineered bacteria capable of degrading environmental pollutants like microplastics.

To better understand the impact and potential of our project, we engaged in multiple conversations with stakeholders, including:

• university professors and agricultural researchers,
• representatives of fertilizer companies,
• and municipal authorities working in sustainability and waste management.

All stakeholder interactions were conducted in full compliance with the GDPR and national data protection legislation. No photos, videos, or identifying information were recorded without documented, informed consent. In cases where consent for public use was not given, individuals' faces were blurred or anonymized, and all personal data were treated with strict confidentiality.

As part of our Human Practices and Education efforts, we designed a series of interactive activities aimed at students, educators, and the general public. Ensuring safety during these events, especially when working with minors or non-experts, was one of our top priorities.

To maintain the highest standards of biosafety and accessibility:

• We never used living GMOs in any of our outreach workshops, demonstrations, or public events.
• Instead, we developed safe analogues and simulations, including food dyes, gelatin-based media, and custom-made digital animations, to effectively communicate biological concepts.
• All materials were non-toxic, age-appropriate, and classroom-safe, and each activity was carefully piloted by our team before delivery.

In our outreach with students, including minors in schools, we strictly adhered to personal data protection regulations:

• All photography and media were captured only with prior written consent from participants or legal guardians.
• No identifying information was shared without explicit, informed permission.
• Faces of minors were blurred or anonymized in accordance with Regulation (EU) 2016/679 (GDPR) and applicable Greek legislation on data privacy.

Through this framework, we ensured that our science education initiatives were not only fun and engaging, but also ethically and legally responsible, inspiring curiosity while safeguarding individual rights.

At Chitinator et al., we believe that ethical awareness must be integrated into every stage of synthetic biology, from design to implementation. From the beginning, we were committed to building a system that is not only effective, but also safe, transparent, and socially acceptable.

We took several conscious steps toward a responsible design:

• All enzyme production takes place within a closed bioreactor, ensuring that no genetically modified E. coli is released into the environment. This setup fully aligns with the principle of containment-based biosafety.
• The final product of our system, a chitinase-rich extract, is devoid of live organisms and can be safely applied to compost or agricultural settings as a bioavailable nutrient source, or as a substrate for other environmentally beneficial bacteria, such as those capable of breaking down microplastics.
• We openly communicated the scope and limitations of our work to all stakeholders and did not overstate the potential of our system. This transparency was central to building trust with both experts and non-specialists.
• We remained mindful of the risks associated with synthetic biology and considered future directions such as enzyme immobilization, kill-switch systems, or modular bioprocess control, not as features we implemented, but as important safety components to be considered if the system is scaled up.

Throughout the design and testing of our system, we remained guided by the belief that synthetic biology must serve the world not only through innovation, but through responsibility, transparency, and respect for the environment and society.