Throughout every phase of the Bye-O-Film design and creation process, our top priority has consistently been safety. Our commitment extends not only to safeguarding the well-being of our team, collaborators, and partners but also to fostering the development of our project in the most ethical and socially responsible manner possible. Within the laboratory setting, health and safety stand as our foremost priorities, forming the basis of a successful project. General safety and safety by design were aspects that we included at each step of the planning. We were certified and educated on the safety requirements of an ML-1 lab. Additionally, we chose to work with safe alternatives when it came to the biological models. This section of our project’s Wiki elaborates further on our conscientious efforts in developing Bye-O-Film.
Introduction
Education
When working in the lab, you must be knowledgeable about lab safety practices. To work in the ML-1 laboratory, a Safe Microbial Techniques (SMT) certificate is required. This is acquired by following the Safe Microbial Techniques (SMT) course. Before the lab work began, all team members who did not yet have the SMT certificate were given the option to obtain it. This ensured that all team members working in the lab were educated and qualified to do so.
Laboratory
We conducted our work within an ML-1 lab, exclusively handling organisms categorized at risk level 1. In the lab, we implemented good laboratory practices to ensure a safe working environment and to prevent genetically engineered organisms from escaping the lab (including general hygiene measures such as hand washing & lab coats, autoclaving, chemical and biological waste bins, and daily cleaning of workbenches). Furthermore, we had access to two autoclaves in the lab, used to process all waste according to university regulations.
Microbes and Biological Safety
For the genetic engineering, we used whitelisted parts (the complete part list is here [Parts]). Additionally, we used the Phage M13, also whitelisted. This phage DNA was separated into a phagemid and a helper phage. Additionally, when working with the phage, we made sure the Virkon was used to disinfect the benches.
Chemical safety
One safety precaution we took in particular was for the gel electrophoresis. When making the gel, a dye is added which is known to be carcinogenic. Conventionally Ethidium Bromide is used, which is known to be carcinogenic and requires high levels of care (wearing 2 layers of gloves to prevent direct contact). Instead of Ethidium Bromide, we used SERVA DNA Stain G which is safer. Despite this, we still took necessary precautions and wore gloves at all times when working with the gels.
Dual Use
Although our project is not harmful and does not generate knowledge with malicious applications, we do introduce design ideas that could
bring up possibilities to individuals with ill intent. Dual-use research of concern involves research intended for clear benefits but that
could be misapplied to cause harm. In this context, the dual-use risks primarily involve the potential misuse of the project's innovative
approach, which combines a genetically modified E. coli-based biosensor and engineered bacteriophages to detect and disrupt biofilms on
medical implants.
The project's biosensor technology, intended for early detection of biofilm formation, could potentially be adapted for malicious purposes,
such as detecting the presence of bacteria in public spaces, which raises concerns related to privacy and surveillance. To address these
dual-use risks, our team adheres to established biosecurity and biosafety guidelines to prevent accidental releases of modified organisms
and is careful about information safety.
In a broader picture, engaging with bioethicists, regulatory agencies, and the broader scientific community will be essential in navigating
the dual-use challenges that could arise if Bye-O-Film is set for further development, beyond the scope of iGEM. By taking these
precautions, the project can maximize its positive impact on healthcare while minimizing the potential for misuse or unintended harm.
Data Protection and Informed Consent
Ensuring safety in our iGEM project extended beyond lab safety, encompassing data protection and informed consent for all stakeholders.
Drawing from Groningen iGEM teams' experiences in 2022 and 2021, we adopted the AREA framework to responsibly collect data while
prioritizing data protection and informed consent. The Groningen teams contributed a valuable informed consent template that guided our
approach. This method involved conducting interviews, summarizing data, and deleting most data at the iGEM season's end, except for an
anonymous summary. To ensure compliance with data protection laws, we sought input and approval from our supervisors, the ethics committee,
and the data security office at our university. In addition, we were in contact with two academic experts from the fields of gerontology,
sociology, psychology, and social and communication studies, who reviewed our survey methodology and survey questions.
Our commitment to responsible data handling and participant well-being adhered to the principles of the European General Data Protection
Regulation (GDPR). We took meticulous steps in data collection, avoiding vulnerable groups and refraining from collecting personally
identifiable information. We also provided stakeholders with comprehensive information sheets alongside our consent form, addressing data
withdrawal, storage, access, and publication. Our customized informed consent sheet adhered to GDPR requirements and aligned with our
research goals, ensuring ethical practices throughout our project.
Biocontainment beyond the iGEM Project
- Is the phage dangerous to humans and can it affect the human microbiome? M13 is not a lytic phage and will therefore not cause cell death [5]. Additionally, the M13 phage only infects certain strains of E. coli. Other bacterial species will not be infected. Lastly, it has been shown in vivo that M13 phages do not influence the microbiome and health of chickens [5]. We do recognize that M13 in combination with Dispersin B may impact the microbiome. This is an area where more research would be required.
- Is Dispersin B dangerous for eukaryotic cells and tissues? Dispersin B is an effective and safe antimicrobial treatment in vivo in multiple animal models [3][4].
- Is Dispersin B dangerous for eukaryotic cells and tissues? Although our experiments are proof of concept and far away from clinical implementation, biocontainment in a clinical setting was an important safety consideration. We planned the design of a sensor that 1. Contains the E.coli and 2. Can release the phages into the site of infection. Some ideas we developed included:
- In the lab, we use plasmids with antibiotic-resistance genes to select the successfully transformed bacteria. If this biosensor were to be tested for real-world use, this antibiotic-resistant gene would need to be removed from the plasmids.
- A porous container with selective permeability. This would allow the diffusion of small molecules like cyclic di-GMP and bacteriophages, but not the bacteria. However, research has shown that e.coli can pass a channel as small as 450 nm [7]. Sterlitech sells Polycarbonate Membrane Filters with a pore size of 400 nm [8]. A material such as this could be used to encapsulate the bacteria but still allow for diffusion of molecules including the m13 phage and the cyclic di GMP [9].
- Using a replication-deficient and/or non-pathogenic/attenuated strain of E. coli would be an option. For example, live-attenuated e.coli vaccines have been produced and found to be safe in humans [10]. Developing a similar strategy for the biosensor would be optimal.
- Lastly, a kill switch should be introduced, for example, a temperature or light-based killswitch, as the 2022 Groningen Igem team, nanobody, explored [6].
A combination of these methods would prevent the escape of the bacteria into the body as well as into the environment and also prevent any damage in case of escape.