Agreement on iGEM safety rules and policies
All research conducted by the iGEM team Munich 2019 was in accordance with the iGEM safety policies and rules. All organisms used and all parts generated by this team are on the whitelist or were approved by iGEM via a Check-In.
Biosafety is the application of knowledge, techniques, and equipment to prevent personal, laboratory and environmental exposure to potentially infectious agents or biohazards.
William C. Shiel Jr.
This year’s team worked at the Helmholtz Centre Munich in the laboratories of the Institute of Developmental Genetics. All work was conducted in biosafety level one (BS-L1) laboratories. BS-L1 is the lowest of four levels and allows you to work with risk group one (RG-1) organisms, which pose no risk to individuals or the environment. Before we started in the lab, all team members received a general introduction to the lab space including the location of emergency exits, eye showers, and emergency showers from our supervisor Susanne Pettinger. This was followed by a more detailed training for responsible work in biological laboratories held by the biosafety officer Dr Jordi Guimera. This training included proper handling of RG-1 organisms and general lab rules like no eating and drinking in the BS-L1 space.
Besides, we were informed about the difference between the biosafety level and risk group and their connection. Furthermore, we were instructed about cleaning procedures in case of spills and the operation of all machines. One point Dr. Guimera emphasised on was the containment of GMOs. To make sure everyone works responsible and all data can be traced back to the experiment all team members are keeping a record in form of a paper lab book and an online lab book as well as a detailed list of all GMOs we are producing during our work. All material that came into contact with our GMOs was autoclaved. No animal, human, or gene drive experiments were performed. All team members were equipped with lab coats, safety goggles, and gloves.
Lab safety
Before we started working on our project in the lab, we did extensive research on the safety aspects of our project. Other than meeting the safety regulations of the Helmholtz Center Munich we made sure to identify risks according to iGEM's definition. At the beginning of iGEM, our team thought of the experiments we need to prove that our project works. Next to the experiments, we also thought of safety aspects and ways we could make sure that our safety as well as the safety of the environment is ensured.
Organsims
The first thing we had to consider was the choice of organism. Organisms are divided into four risk groups, which consider the risk of organisms to the individual working with it and to the community and environment. In accordance with the iGEM safety rules this team only worked with organisms from risk group one with no or low individual and community risk. All cloning was performed using the E. coli strain NEB stable. All experiments were performed with the highly characterised human cell line HEK293T.
Parts
Our project is based on a modular platform. With this system, you can choose a vesicle based on your needs which can be loaded specifically with different proteins and mRNA. For this system to work our team needed to create a lot of different parts. Many of our parts were specifically designed to work for our system and are synthetic, these won't work outside of our system. The parts we use, with origin in an organism, conform with the iGEM whitelist. Only one part we are using originates from a risk group 3 organism: For the formation of our Virus-like-vesicle, we use a modified version of the Human Infectious Virus capsid protein Gag.
HIV's Gag is a polyprotein that mediates viral membrane budding and which gets cleaved by the viral protease into matrix, capsid, and the nucleocapsids. Because the Gag and the Pol genes partly overlap in the HIV genome and can both be expressed through translational frameshifting, there are 204 bp of Pol-coding bases at the 3'-end of Gag. Pol has a total length of 2985 bp. These 204 bp code for the "Gag-Pol transframe peptide", which is 56 amino acids long and activates the viral protease. But neither the coding sequence for the reverse transcriptase nor the coding sequence for the viral integrase is located inside the Gag gene we want to use.
Chemicals
For some of our experiments, we are using chemicals which can be dangerous to individuals. Before using chemicals, we checked the H and P sentences to make sure we handle them in the right way. For example, Trizol, which we use for RNA extraction, evaporates at room temperature and is toxic. Thus, we only use this chemical under the fume hood and with gloves. All spills are immediately whipped off with ethanol.
One point often found on the safety pages of former iGEM teams is the usage of ethidium bromide, which intercalates double-stranded DNA and could thus act as a mutagen. To avoid all risks of ethidium bromide our team decided to use the dye SybrSafe. This dye intercalates well with DNA but is not membrane permeable. Even though this reduces the risk for humans greatly we still use gloves when working with SybrSafe or the stained agarose gels.
Machines
In general, there is always a risk when using electrical devices. To avoid the risk of electrical hazards the Helmholtz Center Munich tests all devices annually. Before starting in the lab all devices were inspected visually for obvious defects like loose cables or broken connections. The operation instructions for each device are on display, in addition, we received an instruction on the proper operation of centrifuges by our supervisor.
Project Design Considerations
To use our vesicles for diagnostic purposes they have to fulfil high safety standards to guarantee the patients’ health. As an iGEM team we can only do basic research on cell models to demonstrate the general applicability of our platform. However, we already considered the ideal properties our cells should have during the planning and research phase.
Cell Viability
Thinking of medicine, patients are amenable to taking pills daily or renewing their vaccination every few years. But transplantation should ideally be done only once in a lifetime since it is a highly invasive procedure. Thus, our cells need to be able to express our vesicles long term without negative effects. To make sure this is the case we performed a viability assay. to proof that our system is not harmful to human HEK293T cells.
Passive Transfection
We have tested that only a small portion of the secreted containers can be taken up by other cells (collateral transfection). To ensure that cell functions of those cells are not affected, the sequence for the gene identifier can be chosen such that it does not encode a protein.
Application Safety
We anticipate ALiVE to be a powerful method to longitudinally monitor engineered cells in cell culture and organoid systems. Of course, any future use of genetically modified cells bears a set of risks that have to be carefullymitigated through controlled processes as has been recently completed for CAR-T cell therapy.
References
Fu-Hsien Y. et al. Gag-Pol Transframe Domain p6* Is Essential for HIV-1 Protease-Mediated Virus Maturation. PLoS One 10, e0127974
Langer S., Sauter D. Unusual Fusion Proteins of HIV-1, Front Microbiol. 9, 2152 (2017)
Solbak et al. HIV-1 p6 - a structured to flexible multifunctional membrane-interacting protein, Biochimica et Biophysica Acta - Biomembranes 1828, 816-823 (2013)
Biohazard Icon. Created by Lukáš Jača from Noun Project.
Medical Definition of Biosafety
Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.
