Team:BGU Israel/Safety
Safe Project Design
In the OriginALS project we worked with bacteria, several different mammalian cell types, and several viral vectors. The bacteria (DH5-alpha) and mammalian cells used in our experiments are organisms classified as biosafety level 1. Meaning, these organisms are safe and do not pose a threat to humans or the environment. In addition, these organisms were handled according to official safety protocols, using appropriate safety equipment, and under the supervision of our advisors.
The mammalian cell lines we used are from the American Type Culture Collection (ATCC): BV2, C8D30, HEK293. All these cell lines meet the relevant safety standards. Mammalian cells require specific handling and storage conditions. Meaning, these cell lines are very sensitive and will not survive outside of the laboratory environment, eliminating any threat they may pose outside of the lab.
In order to minimize the risks associated with bacteria, we limited ourselves to working with vectors which provide resistance to only three different antibiotics (chloramphenicol, ampicillin, and neomycin). In this way we prevented the bacteria from developing resistance to a wider range of antibiotics (any antibiotics we did not use). This way minimizing any risk this bacteria would pose to the external environment. Additionally, we limited ourselves to using only one type of bacteria, DH5a, in order to minimize risks.
All the plasmids we designed, including Crisper-Cas9 constructs, contain a murine promoter, therefore there is little risk of these plasmids being expressed outside of murine cell lines. However, even if somehow these plasmids were transcribed, there is very little risk of this causing a biological hazard since the components of the Crisper-Cas9 constructs are separated into several different plasmids. Only if several plasmids from the same system are transcribed in the same cell will their function be expressed.
Initially we designed a delivery system through the use of Adeno-associated virus (AAV) with serotypes 6 and 9 (AAV6, AAV9). AAV’s are replication-defective (they do not replicate or package additional genetic material) and are therefore defined as non-infectious and non-hazardous. Although we designed and synthesized plasmids for AAV packaging, we did not use these viruses in our experiments. Due to time constraints, the experimental plan was adjusted and we used Lentivirus (2nd and 3rd generation) in order to infect our target cells. Second and third generation Lentiviruses are defined and designed so that the transfer, packaging, and envelope components are separated into three different vectors. Therefore, the lentivirus cannot be expressed unless all of these components are expressed in the same host cell. Hence, second and third generation lentivirus is defined as biosafety level 2; non-infectious, non-hazardous, and safe for laboratory use.
Safe Lab Work
All work in the lab was conducted according to Ben-Gurion University laboratory safety regulations. We participated in several safety training sessions with Dr. Liliana Astachov, which is the university’s nominated biosafety specialist and inspector.
These sessions covered everyday lab conduct (working in a biosafety hood, working with cell lines) and working with viruses. The team also completed an online lab safety course which included an exam.
Initially, not all team members were experienced with genetic engineering lab work, therefore all such lab work was conducted under supervision from an experienced staff member.
Lab safety was an integral part of our ongoing lab work. This was expressed in the following ways:
Personal safety: All lab work was conducted while wearing gloves, safety goggles, closed-toe shoes, and lab coats. When working with viruses, we used additional safety precautions such as using filtered pipette tips.
Clean and tidy work environment: all work benches were cleaned and disinfected with ethanol at the beginning and end of each work day. We separated our waste into four categories: viral waste, biological waste, chemical waste, and non-harmful waste. Each of these types of waste was disposed of after autoclaving. Viral waste was incubated with bleach, exposed to UV radiation for twenty minutes and only then processed in the autoclave along with biological waste.
Separation of the work space according to the type of work: The first area was designated for ongoing activities such as procuring plasmids, Gibson assembly, restriction and ligation experiments, midi-prep, etc… The second area was a chemical hood where we conducted gel electrophoresis analysis using Ethidium Bromide. We also worked in the chemical hood when using hazardous liquids such as isopropanol. The third area was designated for work with mammalian cells and viral vectors. This area was secluded from the rest of the lab in order to guarantee safety and prevent contaminations. This work area included a biological hood, a sterile incubator for mammalian cells, and other tools designated only for work under sterile conditions. The following procedure was followed each time we worked with the cells: the hood was sterilized using bleach and ethanol before being exposed to UV radiation for at least twenty minutes (at which point team members were not allowed in the room). All work with the cells was done with sterile tools and repeated sterilization of one’s gloves with ethanol.
Safe Shipment
Only DNA parts were shipped and properly documented. We did not experience any safety issues when shipping our DNA parts.
