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General laboratory safety practices

During the first weeks of work in the lab, the team attended a workshop on biosafety and good laboratory practices. During this session the team members had the opportunity to practice correct pipetting, safe removal of gloves, and correct labelling and handling of dangerous chemicals that should be handled in the fume hood at all times.

Figure 1. Fire extinguishers

The team was offered the opportunity to have training on waste sorting and management by Uppsala University. During this training we were taught how to safely label and dispose of material that had been contaminated with either chemical or biological matter. Recycling was also a major topic during this training session and we were taught how to sort packaging from several lab supplies such as colored glass bottles, laboratory glass, electronic waste, regular paper or cardboard boxes and plastic packaging.

Moreover, all members were introduced to the laboratories where we would be working on our project. All first aid stations, eye showers, and showers were shown to the team along with instructions on how to use these in the appropriate circumstances. The team members were also taught how to use fire extinguishers and which kind of fire extinguisher is indicated for different kinds of fire sources. We were also taught how to use a fire blanket and how we should proceed in case of a fire. This included pointing out the location of the emergency exits and also the meetup point outside the building.

Figure 2. Autoclave

The lab coordinators were introduced to how to correctly and safely use the autoclaves and other equipment used for cleaning and sterilizing lab material. The team also spent some days introducing all the members to basic laboratory work such as aseptic techniques, transformation, PCR, and agarose gel electrophoresis. All gel preparation, loading and electrophoresis were performed at designated stations in order to keep the handling of DNA dyes, such as SYBR safe, restricted to specific benches.

The E. coli strains

Figure 3. Bunsen Burner

The two strains of E. coli , that we work with in a BSL1 laboratory to construct various plasmids (DH5alpha) and express the proteins (BL21 DE3), are non-pathogenic and would quickly be outcompeted by natural bacteria if they were to be present in the environment. This is because, in nature, the optimized conditions that they experience in the laboratory are very unlikely to be achieved.They are also included in the iGEM white-list.

However, the plasmid for expression possesses a kanamycin selection marker which led to us having to use this antibiotic to screen for transformants in the several cloning steps that our project required. If some kanamycin was to be accidentally released into the environment, it could certainly contribute to the increasing number of antibiotic-resistant bacteria which are a very pressing health concern [1]. At a point in our project we also used E. coli BL21 DE3, which contained a pLysS plasmid with a gene for chloramphenicol resistance. This meant that, in order to keep that plasmid, we had to use chloramphenicol which can have the same effects as kanamycin if leaked into the environment.

Therefore, we worked carefully with these antimicrobial substances and disposed of them safely, by following the guidelines that were taught to the team. This was done to avoid any spills or leakage. The cells and all the glassware the team used were treated with Jodopax to kill the cells and avoid possible leakages into the environment.

The growth factor

Figure 4. Common hazard symbols

The FGF2 growth factor that we optimized is a mammalian protein involved in cell signaling [2]. This growth factor is quite fragile and is not active for too long even when inside an animal’s body, which means that it would be rendered inactive even more easily, by the harsh conditions outside an animal’s body [3]. Even if it could remain active for a reasonable amount of time under these conditions, this growth factor binds specifically to its host’s FGF receptors [4] which means that it is very unlikely to affect other forms of life.

Even though the risk is low, we always worked in the safest way possible to minimize the risks of leaking this growth factor into the environment.

Mammalian cell cultures

Figure 5. Laboratory waste

The mammalian cell culture was carried out in a BSL2 laboratory inside a laminar flow hood. The cells themselves could present themselves as a potential source of infection if they were to be contaminated by human or animal pathogens during culturing. These pathogens could reach other individuals if the cells were not disposed of safely.

Therefore, we always worked while following the university’s guidelines and safely disposed of contaminated material and mammalian cells in order to avoid possible leakages into the environment.

COVID-19 Measures

During the spring of 2021 all team meetings were held digitally to follow Uppsala University’s guidelines. Fortunately, during the summer months the team received an exemption from digital teaching which allowed us to work in the lab. However, during these months, guidelines such as physical distancing, mandatory face mask use in the lab, and regular disinfection of hands and surfaces were enforced to contain and minimize the chances of an outbreak. All team members were advised to stay home if they felt the slightest symptom and to get tested as soon as possible. Moreover, all team members had the option to virtually attend all the meetings held. Throughout the project the team worked following the guidelines from the Swedish Health Agency.

References

[1] “Antibiotic resistance.” https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance (accessed Sep. 20, 2021).

[2] D. M. Ornitz and N. Itoh, “The Fibroblast Growth Factor signaling pathway,” Wiley Interdiscip. Rev. Dev. Biol., vol. 4, no. 3, pp. 215–266, Jun. 2015, doi: 10.1002/wdev.176.

[3] G. Chen, D. R. Gulbranson, P. Yu, Z. Hou, and J. A. Thomson, “Thermal Stability of Fibroblast Growth Factor Protein Is a Determinant Factor in Regulating Self-Renewal, Differentiation, and Reprogramming in Human Pluripotent Stem Cells,” Stem Cells Dayt. Ohio, vol. 30, no. 4, pp. 623–630, Apr. 2012, doi: 10.1002/stem.1021.