Safety

While we believe that synthetic biology has the potential to improve the lives of many, we are also aware that engineered organisms could bring harm either through unintended consequences or through intentional misuse. In particular, synthetic biology research poses a risk at three main levels: (1) to the individuals working with the materials; (2) to public health if organisms are released beyond containment; (3) to the environment if organisms are released beyond containment. It is therefore crucial to consider safety throughout every stage of the project - from experimental design to day-to-day activities. A useful framework for this is the hierarchy of controls. This page details the decisions we took over the course of our project to minimise the risk.

Elimination

The most effective way to reduce risk is to eliminate the hazard from the experimental design entirely. This is something we started early on in our project, realising that many of our initial ideas were not feasible to do safely with the resources and expertise available to us.

Inverted triangle split into five layers. Each layer has a term, elimination, substitution, engineering controls, administrative controls, and ppe. This image has elimination in a different colour to the other layers.

Magnetic Nanoparticle Synthesis

We initially considered synthesising the magnetic nanoparticles required for our system ourselves. However, the protocol we considered for this involved high pressures and temperatures, as well as requiring the exclusion of oxygen from the reaction mix (Liang et al, 2023). Ultimately, we decided that it was beyond the existing expertise of our team members, and while we may have been able to receive training from the Chemical Engineering and Biotechnology department, it was not core to our project, and so we decided to order our magnetic nanoparticles commercially instead.

Substitution

Where elimination is not possible, the next most effective method for risk management is to swap the hazard for a safer alternative. This might be through reducing the concentration of a reagent or using it in a different form.

Inverted triangle split into five layers. Each layer has a term, elimination, substitution, engineering controls, administrative controls, and ppe. This image has substitution in a different colour to the other layers.

Choosing model organism

Since our system involves the interaction of magnetic nanoparticles with cell surface proteins, we initially thought that animal systems - such as C. elegans - which don’t have cell walls would be ideal. This would also have allowed us to work with human proteins that have already been shown to work with magnetogenetic systems, as expression of mammalian cell surface proteins is not very transferable to microorganisms such as yeast and bacteria. However, we ultimately decided that it was not possible for us to work with such organisms safely. Deciding this early on allowed us to switch our focus during the literature review stage to look for pathways in S. cerevisiae and E. coli that could be manipulated for magnetogenetics, as opposed to human proteins that have been characterised in magnetogenetics.

Chemiluminescence Calibration

When researching protocols to calibrate our chemiluminescence measurements, we noticed that most procedures used a 30% hydrogen peroxide solution to oxidize luminol. However, hydrogen peroxide at this concentration poses significant safety risks, including severe eye damage and harmful effects on aquatic life. After conducting calculations, we determined that using the same volume of a 3% hydrogen peroxide solution would still provide an excess of oxidizing agent for the luminol reaction. This means that 3% hydrogen peroxide can be used effectively while significantly reducing the associated risks, making the process much safer.

Engineering Controls

Engineering controls refers to the “safety by design” approach, which aims to minimise the risk posed by human error/malicious intent by engineering out possible harms. This is often done by physically separating people from dangerous equipment.

Inverted triangle split into five layers. Each layer has a term, elimination, substitution, engineering controls, administrative controls, and ppe. This image has engineering controls in a different colour to the other layers.

Isolation

All experiments were carried out in a BSL-1 lab, with biosafety cabinets being used where needed for extra sterility. Some experiments required us to use equipment at both the Department of Engineering and the Department of Genetics. When moving samples between the two, the media containing E. coli and S. cerevisiae was sealed in hermetically closed tubes that were placed in a sealed bag containing sufficient absorbent material to mop up any liquid spill. The containers were labelled, showing that they contain GM E. coli or S. cerevisiae in hazard group 1. The samples did not require temperature control during transportation.

Domestication and disabling of strains

S. cerevisiae and E. coli are good candidates for model organisms from a safety perspective, since they are very commonly used and so standard method exist to minimise the risk posed by accidental risk.

For E. coli strains, this is through domestication. The strains we used were as below:

  • BL21(fhuA2 [lon] ompT gal [dcm] ΔhsdS)
  • DH5α(fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17)

For S. cerevisiae strains, this is through auxotrophic mutations that make them much less resistant to harsh environments than wild type strains. We also filled in the check-in form, as S. cerevisiae is a spore-forming fungus not on the iGEM white list. The strains we used were as below:

  • YPH499 (MATa ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1)
  • HAS100L (MATα ura3-52 leu2-3,112::pAS17L(2x) his3-11,15 MAL SUC GAL)
  • Y1 (wsc1::KlURA3 leu2−3,112)

Administrative Controls

Having established procedures around safety allows institutions to ensure thorough and consistent risk management for all individuals working there.

Inverted triangle split into five layers. Each layer has a term, elimination, substitution, engineering controls, administrative controls, and ppe. This image has administrative controls in a different colour to the other layers.

Training

Our formal training had three main components: (1) Watching a 35 minute presentation on the Management of Biological Safety at the University of Cambridge; (2) Working through a presentation and quiz on chemical and laboratory safety; (3) Attending a biosafety session with Dr. Thierry Savin, where he went into detail about the department’s policies around safety in bioengineering.

Risk Assessment

We also submitted a risk assessment to the Department of Engineering. This included:

  • A detailed overall risk assessment describing training, planned experiments, waste disposal and decontamination procedures, and out of hours/lone working policies
  • A form specifically detailing the biological risks, describing the strains, vectors, and genes used, in addition to the experiments we planned to do with them
  • COSHH forms
Completing these documents encouraged us to think carefully about the risks associated with our reagents, hardware, and strains. It also gave us a simple set of things to refer to when unsure about things like waste disposal.

We also submitted the iGEM safety form, which helped us solidify our understanding of the risks in our project, as well as the risks that may arise if our project was pursued further. You can find our full safety form at this link: iGEM safety form

PPE

The final level of risk management is relying on individuals to protect themselves and others. In particular, this involves wearing lab coats and gloves, tying up long hair when working with a flame, and washing hands before leaving the lab.

Inverted triangle split into five layers. Each layer has a term, elimination, substitution, engineering controls, administrative controls, and ppe. This image has PPE in a different colour to the other layers.

References

Liang, Y., Jiang, L., Xu, S., Ju, W., Tao, Z., Yang, Y., Peng, X., Wei, G., 2024. Synthesis and Characterization of Fe3O4 Nanoparticles Prepared by Solvothermal Method. J. of Materi Eng and Perform 33, 6804–6815. https://doi.org/10.1007/s11665-023-08431-1