Introduction

Working in a safe and secure environment is an essential component of responsible innovation and research. By continuously identifying and managing possible risks, we try to minimize hazards to our iGEM team, university colleagues, society, and the environment. Therefore, we consider the whole picture from physical and lab-related risks to project design safety in our safety approach. This page will discuss the safety measures we took during the design and experimental phases of !MPACT.

Lab safety

All research conducted by the iGEM team Eindhoven 2022 is in accordance with the 2022 iGEM safety policies and rules.¹ Most of the organisms and parts used and/or generated are on the iGEM 2022 White List. Parts that are not on the White List, are successfully submitted via a check-in form before usage. These are IL-10, HEK 293T cells and anti-PR3 antibodies. Throughout the whole development of !MPACT, all national and institutional rules and regulations are strictly followed.²

General Safety

Safety preparations before lab work

As this year's iGEM team is working partially in an ML-1 and ML-2 classified laboratory (equal to biosafety levels 1 and 2, however only ML-1 work is performed), all of the team members had to follow multiple introduction sessions given by the lab technician of each lab. These introductions elaborately covered safety policies and rules inside the specific labs. The location of all safety equipment was shown during this introduction (Figure 1).

Figure 1 | Safety supplies in the lab. Upper left is a CO₂ fire extinguisher. Upper right is a stop button for electricity. Bottom left is a fire blanket. Bottom right is an eye wash.

Moreover, before we got granted access to each lab, we successfully passed an exam about safety in both labs.

During the design process of our project, we closely collaborated with the Biosafety Officer (BSO) of our university. We not only discussed the safety of our project in the lab but also the safety of our project with regard to society and the environment. The latter is described on the safety page which can be found by clicking on this link. Furthermore, each team member followed, and successfully passed the Safe Microbiological Techniques (SMT) course given by the Biosafety Officer, granting every member the SMT certificate.

Finally, we got an introduction to all the lab equipment that we are using. These introductions are given by the Ph.D. student or Postdoc who is responsible for the maintenance of the equipment. Possible hazards are always discussed with our lab supervisors.

Figure 2 | Jolien working safe in the lab. Jolien is working in the ML-I lab, wearing a labcoat, gloves and glasses. Futhermore, her hair is tied.

Safety measures during lab work

For personal safety, during lab work, lab coats and lab goggles are always used. Long hair is tied up and depending on the experiment, disposable gloves are used. (Figure 2)

Transport of materials and chemicals between labs is done appropriately by using each specific containers.

Research conducted on mammalian cell lines is done inside the Cell Lab on our campus. Here multiple laminar flow cabinets are present which are applicable for cell work on cells categorized in ML-2 (including HEK 293T cells). (Figure 3)

Figure 3 | working safe in a ML-II lab. When working in a ML-II lab all the regulations for the ML-I lab count. Furthermore when performing an experiment you have to work in the biosafety cabinet. Picture taken by Bart van Overbeeke Photography for Eindhoven University of Technology.

Biological safety

Vectors and IDT gBlocks

For our project, we used multiple vectors, of which detailed information can be found on the Parts wiki page. By making different insert variants in pLeo619 we were able to achieve a small library of membrane receptors, each with their own linker length. Moreover, we used pSB1C30 to express GFP and UTR+GFP, for Part improvement. pLS13 is used as a mammalian reporter plasmid for STAT-3-induced SEAP expression. pLS15 is a mammalian STAT3 expression vector.

Bacteria Strains

For our research, we have used different E. Coli strains. We used TOP10 Chemically Competent cells (NEB) for transformations of difficult ligation and Gibson assembly products. For more general transformations we used DH5α Subcloning Efficiency (ThermoFisher), XL10-Gold Ultracompetent (Agilent), NovaBlue (DE3) (Sigma Aldrich), and BL21 (DE3) (ThermoFisher) cells. All the cells have been altered to be safely used in an ML-1 lab. All GMO-based lab work done on bacteria was registered in the GMO notebook present in the laboratory.

Mammalian Cell lines

For all of the mammalian cell work, we have used HEK 293T cells. As this cell line has inconsistencies in the risk group classification, we have contacted iGEM’s Safety and Security Committee. This communication led to the conclusion that the cell line is categorized in Risk group 1 in the Netherlands.

Human Practices Safety

“The safety of people shall be the highest law”- Marcus Tullius Cicero

Ethical & Responsible Innovation

As an iGEM team, we highly value ethical responsible research with human subjects. We carried out our research with diligence and according to the Netherlands Code of Conduct for Research Integrity. ¹

Our research is tested according to the Code of Conduct by the Ethical Review Board Committee (of Eindhoven University of Technology (TU/e)) from whom we received official institutional approval to conduct research on Human Practices involving human subjects. The Ethical Review Board Committee helped us prepare the informed consent forms to make participation in our research comply with Dutch law and to protect the participants of our research. A data steward our university helped us secure the sensitive and personal data of the participants and reviewed our PIA (Privacy Impact Assessment) to ensure we meet the requirements of the General Data Protection Regulation (GDPR) and the Algemene Verordening Gegevensbescherming (AVG) which is the Dutch law concerning the privacy of personal data. Moreover, we took it one step further, since we believe that building a good relationship with the stakeholders benefits their participation. This we achieved by sharing our enthusiasm and achievements with them. We learned that this resulted in more enthusiasm among our partners, as they feel part of our project design process.

Here you can view the informed consent form for patients we engaged as an example. It contains information on which data we gather, how we process it, and how we store it responsibly to ensure the safety and privacy of the participants.

Safety during the challenge day

On the 14th of September iGEM team Eindhoven organized the challenge day for high school students. One of the exercises during this day was painting with fluorescent bacteria. According to the legislation in the Netherlands a GMO certificate is needed when you perform lab work with GMOs. The laboratory we used for this experiment was different from the laboratory we usually work in. Therefore, this certificate was arranged. A GMO certificate was applied for ML-1 activities, for the laboratory we would be working in for that specific day.

Besides the GMO certificate, safety was also ensured during the day. In the laboratory the students got a small safety instruction in how to work in the laboratory. This safety instruction was provided by the biosafety officer. To help with the exercises tutors were needed, for the E. coli art 1 tutor for 4 students was presents for all the other exercises 1 tutor per 10 students. Furthermore, all tutors were asked to set the alarm number of the university into their contacts on their phone.

Netherlands Code of Conduct for Research Integrity. doi:10.17026/dans-2cj-nvwu

Safety