Team:TU Darmstadt/Safety

TU Darmstadt

Safety

While working on our project safety was an important factor. We strove to minimize risks to members of the team during laboratory work and also took measures to ensure that no harm would be done to the public or the environment.

Safe Project Design

The goal of our project was to develop a toolkit which would ideally be used in many different use cases for Virus-like particles (VLPs). Some applications like targeted drug delivery or vaccinations would ultimately be used on human patients even though at the moment our system is limited to lab use only. This means that the VLPs we are using need to be safe to be injected into a human body if such developments are ever to be pursued. For this purpose we performed endotoxicity assays at Paul-Ehrlich-Institut (PEI) in order to start to get an understanding of the efficacy of our Virus-like particles and whether further steps need to be taken during the purification process. Endotoxin levels of P22-VLP produced in vivo and in vitro were measured via Limulus amebocyte lysate (LAL) test after ultracentrifugation purification.

Figure 1: Endotoxin levels in endotoxin units/ml (EU/ml) and pg/ml in set volume of ultracentrifugation sediment.

In general, the results (Fig. 1) showed that the endotoxin levels in in vitro assembled VLPs were significantly lower than the ones produced in vivo. This was to be expected since in vivo samples are known to hold higher amounts of endotoxins because of their bigger risk in contamination from the bacteria during cell lysis. Nevertheless, this concentration is still remarkably high. In conclusion more purification is needed so they fit the medical standards. We are confident that this can be achieved as other medically relevant agents are also produced in E. coli such as human insulin. It can also be observed that unmodified VLPs showed slightly higher endotoxin amounts than modified ones. This however is most likely due to an insufficient purification process rather than showing that modified VLPs could inherently be more endotoxin free.

In the case of the vaccination application one advantage that VLPs inherently present is that they are not infectious but still cause an immune response.[1][2] Whereas vaccines based on inactivated pathogens may have side effects[3] VLPs have the potential to be relatively more safe in this regard since they do not contain viral DNA.[1] This also means that in the production of such vaccines work with pathogens would not be necessary, reducing the risk of infection in people working in vaccine production.

The enzyme we are using for the modification of our capsid originates from Staphylococcus aureus which belongs to risk group 2.[4] To avoid working with this organism we decided to express the Sortase A7M in E. coli as the enzyme itself does not pose any risk to human health. It is neither toxic in itself nor does it catalyze any toxic reaction.

A regular worry in any iGEM project is dual use. In an effort to anticipate and avoid any potential of using our project in harming people we talked to Prof. Susanne Bailer who works on VLPs at the university of Stuttgart. She told us that there generally lies no threat to human health in VLPs and that she does not see dual use as a primary concern of our project.

In his 2008 article[5] M. Schmidt brings attention to the fact that any step forward in synthetic biology, that makes future development easier, could also cause safety problems. Simplifying the technology potentially allows users who are not proficient in the field to work on their own molecular machines. This "diffusion", as he calls it, could lead to synthetic biology being so readily available to laymans that it could become hard for safety standards to be upheld. With our project directly contributing to this diffusion we feel it our responsibility to mention this fact and urge any other iGEM teams to keep in mind the possibility of their project being used by hobby-biologists and if this calls for additional safety measures.

As we questioned people in public during the "Hessentag", an annual cultural event in our region, a common factor among all those we talked to was their concern for safety in biotechnology and synthetic biology. Among non-scientists especially genetic manipulation is still often perceived as threatening. We believe it as part of our task to spread awareness and reinforce the positive work ethics that will hopefully lead to a change in perspective in the future. In response to this we approached Sibylle Gaisser Professor of Ethics and Biotechnology at University Ansbach who advised us to devise a safety form that could help in preventing misuse. For more information see our integrated Human Practices page or our big report.

Safe Lab Work

Since most of the team members originated from a background in biology or chemistry, the majority of us had worked in a laboratory before participating in iGEM. Nonetheless everyone took part in a day long instruction course and safety training carried out by our primary principal Investigator (PI) and student instructors. An introduction to any machinery that we would be using (e.g.: thermocycler, gel-imaging equipment, centrifuge, …) was given and hazards like gel-staining chemicals, UV-light, acrylamide or Bunsen burners were explained.

We worked in the laboratories of Heribert Warzecha and Viktor Stein (protein purification) at TU Darmstadt both of which fall under biosafety level 1 (S1) meaning no pathogenic organisms were used. Standard equipment while working consisted of gloves and labcoats as well as safety goggles when handling more dangerous chemicals. Two cell lines of E. coli were used: TOP10 for cloning and BL21 for protein expression. Both of these bacterial strains are so colled "safety-strains" in accordance with the GenTSV, the German law for genetic security. [10] . In general all safety regulations required by German law were upheld. Any gear that came into contact with E. coli was sterilized before disposal to prevent any bacteria especially genetically modified (GM) ones from escaping the lab.

Although the genes we modified our E. coli with should neither allow them to harm humans nor spread uncontrollably it was still paramount to keep all GM bacteria confined to the laboratory as they carry antibiotic resistances that were used for selection. As the spread of antibiotic resistant microbes keeps being an unresolved humanitarian issue [6] it was critical to not allow any of the resistance genes we used to be released into nature where they could be transferred to pathogenic bacteria. [7] This was deemed crucial because the antibiotics we used are included as highly important (chloramphenicol) and critically important (ampicillin, kanamycin) on the WHOs List of "Critically Important Antimicrobials for Human Medicine". [8] Every instance of genetically altered organisms being introduced into the environment also represents a possible disruption of natural systems and it cannot possibly be anticipated how they interact with ecosystems they are introduced into. [9] We kept records on all genetically modified Organisms (GMOs) that we worked on and stored for the purpose of full transparancy as well as allowing for a faster reaction in case of a confinement breach.

As a closing remark we made a simple checklist for all future iGEMers to keep to safety standards:

  • Keep the laboratory clean
  • Avoid taking modified microorganisms out of the lab
  • Wear a lab coat, gloves and safety glasses
  • Wash hands before leaving the lab
  • Every person who works in the lab get a safety instruction and a lab introduction.
  • Record the working steps and which GMOs are stored in your lab

References

  1. ↑ Chackerian B., Virus-like particles: flexible platform for vaccine development, Expert Review of Vaccines, 2007, 6(3): 381-390 [1]
  2. ↑ Lowy et al, Safety and Immunogenicity Trial in Adult Volunteers of Human Papillomavirus 16 L1 Virus-like Particle Vaccine JNCI: Journal of the National Cancer Institute, 2001, 93(4): p 284–292 [2]
  3. ↑ U.S. Department of Health and Human Services, Update; vaccine side effects, adverse reactions, contraindications, and precautions : recommendations of the Advisory Committee on Immunization Practices (ACIP), MMWR. Recommendations and reports, 1996, 45(12) [3]
  4. ↑ Public Health Agency of Canada (PHAC), Staphylococcus aureus Material Safety Data Sheet, viewed 10/16/2019,10:56 [4]
  5. ↑ Schmidt M., Diffusion of synthetic biology: a challenge to biosafety, Systems and Synthetic Biology, 2008, 2(1-2): p 1-6 [5]
  6. ↑ Ventola CL., MS, The Antibiotic Resistance Crisis Part 1: Causes and Threats, PT., 2015, 40(4): p 277-283 [6]
  7. ↑ Gopal Rao G., Risk Factors for the Spread of Antibiotic-Resistant Bacteria, Drugs, 1998, 55(3): p 323-330 [7]
  8. ↑ World Health Organization, Critically important antimicrobials for human medicine - 5th rev. Geneva, 2017 [8]
  9. ↑ Snow AA. et al., Four steps to avoid a synthetic-biology disaster, Nature, 2012, 483 [9]
  10. ↑ Verordnung über die Sicherheitsstufen und Sicherheitsmaßnahmen bei gentechnischen Arbeiten in gentechnischen Anlagen (Gentechnik-Sicherheitsverordnung - GenTSV), viewed on 10/21/2019; 01:07 [10]
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