iGEM Athens

SAFETY

Laboratory Safety

General Laboratory Safety

Our team was fortunate enough to have limited access to laboratory spaces, which are equipped with an arsenal of equipement, enough to ensure a safe laboratory experience, void of any danger, to the Wet Lab members, to our colleagues, and the Environment. Even though our wet lab did not produce any results, mainly due to the very short period of time that it was available to us because of the nationwide regulations due to the pandemic, safety was a very important aspect of our practices from Day 1.

We underwent thorough Safety Training, which included proper microbiological aseptic technique, waste management, as well as accident management. We were introduced to the proper use of the equipment we were going to be using, and the dangers of various commonly used chemicals were emphasized upon, as well as ways to manage possible accidents and spills.

Laboratory dress code was strictly followed, which included closed shoes, full length trousers, and mandatory use of a lab coat, not only when doing experiments, but also when spectating. The lab coats were always kept in a drawer located inside the lab, as to ensure no contamination. This minimized any risks of hazard to ourselves, and any accidental release of microorganisms in the environment.

We were very particular about our waste management, as this is often a way in which microorganisms or toxic chemicals are released to the environment due to laboratory experimentation. We made sure to never discard any living microorganisms, and all chemicals were discarded according to the manufacturer’s orders, and local regulations.

Microbiology

Microbiological assays played an important role in our very short stay in the lab. This is why we were very particular and proactive regarding such protocols. An aseptic technique was always used, both to ensure the reproducibility of our experiments and the containment of microbiological material. All of our microbiological work was done in a Biosafety Cabinet, equipped with HEPA filters, that ensure no release. All the work stations were sterilized with 70% ethanol both prior to and after each experiment. Nitrile gloves were worn when working with microorganisms.

We were also very particular as to no live microorganism being disposed of improperly. Our used petri dishes were all autoclaved before discarding, and all liquid cultures were incubated overnight with a strong chlorine/soap solution, before disposal.

Safety also played a vital role in our selection of chassis and organisms used, all of which were deemed BSL Level 1. The main organism used for plasmid propagation was E. coli DH5α. This is a very common engineered laboratory microorganism, with no risk of human pathogenesis.

The strain used for the bacterial conjugation was E. coli S17-1, a very common strain used for this kind of technique. Regarding the structural colouration, the Flavobacteriia strains used were the model organism, Flavobacterium johnsoniae, as well as a strain and a mutant derived from it, named F. IR1, and F. IR1 M16. These are reported to give very strong and vibrant colouration and thus were a very good match to our project [1]. All of the above organisms are regarded as BSL Level 1, and thus are prime candidates to become our chassis. One consideration we have regarding the whole of the Flavobacterium genus was that there are reports of certain species in the genus that are known pathogens to fish [2]. Although this pathogenicity is not known in the aforementioned strains, they were treated as if it were, mainly because the lack thereof is not proven yet. We had to take extra care to manage waste and ensure that no viable cells were released to the environment, as per the standard protocols.

Molecular Biology

Our project, as any synthetic biology project, requires molecular manipulations, that do not come risk-free. Most of the risk harboured by genetically modified organisms is, however, mitigated by our very careful manipulations to ensure No Release. Another risk in molecular microbiology is the use of antibiotics, and the genes that confer resistance to them. Special precaution is taken to make sure that all antibiotics used are not the most clinically significant, and also that the transfection with the resistance genes is done in a controlled environment, only to the designated recipient strains. Our conjugation system has the potential to transfer antibiotic resistance to many species, due to the universality of the S17-1 strain. The conjugation protocol would thus only take place if and only if we could verify that the recipient is the designated Flavobacterium strain. Every modified cell would also be disposed as mentioned above.

Project Safety

General Project Safety

Our project has the potential to be scaled up to manufacturing scale, due to its biological nature, and modular design. In such a scale-up, a factory would produce large quantities of the cell-free, structurally coloured cellulose material, for many different applications.

This implementation, however, does not come without its own risks that have to be dealt with, regarding the environment, the producer, and the final user of the product.

Environmental Safety

The environmental aspect of the project was a main concern of ours, since its inception. We seek to create an alternative to chemical pigments that is a much more environmentally friendly alternative. As stated at the problem section of the project description, the pigment industry places additional strain on the already ailing environment. Our proposed solution, a biological means for production of colour has many advantages, but precautions must be taken.

For one, we propose big scale microbiological cultivation, which can be very dangerous in case of an accident. Though release of a big quantity of genetically modified organisms could potentially have many and diverse ecological repercussions, the fact that our chassis is a BSL Level 1 organism largely mitigates this risk .

Our choice of cellulose has another environmental advantage over commonly used chemical pigments, and that is its undemanding biodegradation. As cellulose is a ubiquitous substance in the natural environment, owing mainly to its abundant biosynthesis by plants, many unique biodegradation pathways have evolved. Many different Fungi and Bacteria have an arsenal of enzymes that convert the polysaccharide into its component mono- and di- saccharides. Those are in turn used as a reduced carbon source for a whole array of diverse micro- and macro- organisms. Also, many biotechnological advances have shaped the field of cellulose degradation, as it is often a cheap and abundant energy source for many biotechnological applications. All of the above can be used in the degradation of the final product, once its intended use period is over.

This objective was further reinforced when we further researched on the complexity of the problem we attempted to address. The threat that synthetic dyes pose both to our health and the planet made us consider human and environmental well-being as a whole and thus propose a solution that serves their needs equally. This brought a dual perspective of safety in our project. We envisioned an end-product that is safe not only for its end-users, but also the environment it is hypothetically released into.

Producer Safety

Another of our main concerns was the safety of the manufacturing procedure, and mainly the risk to which a possible producer is subjected, either be it in a small scale setting (a scientist) or in a big scale one (a worker in a factory). The risk of working with microorganisms is always present, and special precautions should always be taken. With that in mind, both in the laboratory and the factory environment the already mentioned rules must always be closely followed.

User Safety

The final step, and unambiguously the most important is the use of the final product. Our designed material should be as safe as possible to the end user. This was mainly ensured mainly by two factors in our design. The first one has to do with the proposed matrix of our material, cellulose. As the biopolymeric constituent of many everyday items, such as paper and wood, it is generally considered safe. It is biocompatible, meaning that it is not toxic, and allergic reactions to it are rare. The second factor is our choice to opt for a cell free material. The bacteria used to produce it, although considered safe, are inactivated before extraction of the final product. The viability will be tested thoroughly, as to minimize any unforeseen risk.

Taking the above precautions should lead to chemically pure and biologically inert material, that retains all the advantages of a colourful substance.

Team:Athens/Safety