There is a certain element of risk in anything you do, but the potential risks in a molecular biology lab are greater. A molecular biology laboratory is a unique environment that requires special practices and containment facilities, in order to properly protect people working there. This year we journeyed in iGEM's competition placing the safety of ourselves and others at the forefront of our project. We must not forget that safety in the laboratory is the primary concern!
Lab Safety
This year, for our experiments' implemenattion, laboratories classified as biosafety level 1 (BSL-1) were employed. Before starting the experimental procedures in our lab, we attended a training course regarding the lab safety rules and guidelines by Dr. Papadopoulou Lefkothea, Professor of Molecular Pharmacology, which is responsible for maintaining the safety rules of our laboratory. During this training, we learned more about thesafety rules of our lab,the hazardous reagents and procedures and waste management. We talked about the general guidelines for keeping safety at our experimental procedures such as the necessary personal protective equipment and possible risk associated with instruments usage in our biosafety Level 1 laboratory. Furthermore, considerable care was given in the management of possible accidents or emergency situations ensuring the health and safety of all the wet lab members of our team. During this conversation, we developed a well-structured safety list which was applied by all team members during all our experiments:
- Every member was equipped with protective equipment consisting of a lab coat, surgical masks, and safety goggles. Disposable gloves were available at every working station.
- Special attention was given to how tohandle hazardous substances. More specifically, ethidium bromide is a known mutagen, commonly used during gel electrophoresis. All the members were informed on the appropriate handling procedures that should be followed in cases of spills or skin contact. The persons using the ethidium bromide were working on specific fume hood with personal protective equipment to reduce the exposure and enhance their protection. All the products with ethidium bromide contamination were disposed of in specific waste according to the disposal protocols. Acrylamide is also a hazardous substance since it is mutagenic and may cause neural toxicity. When the members of our team were using acrylamide during the SDS gel construction, their health care was ensured, following the manufacturer’s safety data sheets and the appropriate disposal steps. Also, some antibiotics which are used during the cloning experiments for the selection procedure have some hazardous effects at high concentrations. Specifically, ampicillin, chloramphenicol and kanamycin in chronic or acute exposure can be harmfull according to the material safety data sheets (MSDS). Therefore, the team was wearing personal protective equipment (PPE) when using these antibiotics. All the members were informed on the appropriate handling procedures that should be followed in cases of spills or skin contact and followed the disposal protocols.
- Special attention was paid to the use of UV- light during the DNA imaging of the agarose gels. We were instructed to wear personal protective equipment and especially protective UV-glasses.
- We learned the emergency protocols when an emergency occurs, for example, a fire outbreak. In this case, we must know how to use the fire extinguisher, evacuate the lab and immediately call a supervisor for help.
Furthermore, we were trained on good microbial techniques by more experienced members of our lab, to reduce the risk of accidents. More specifically, we were trained in sterilization techniques when working with bacteria and in cleaning and disinfecting protocols in case of an accident. We had a constructive discussion with other experienced members of our lab about the way of handling the certain bacteria that we were going to use. Finally, we were informed about the individual's responsibility for safety in our institution and some of the essential procedures to secure personal information.
Safe Project design
In order to reduce the risks associated with our project and experimental procedures, we consulted our Principal Investigator and Advisors when designing the experiments. In addition, we received another training course by Dr. Papadopoulou Lefkothea, focusing on the risks arising from the experiments that we were planning to perform.
Microorganism information
Since our project is focusing on the construction of an in vitro diagnostic (IVD) test, it does not require any microorganism in its final form. For the experimental procedures we decided to use safe, common laboratory bacterial strains. Given that we only use bacterial microorganisms as hosts for the cloning experiments and the recombinant protein production, we decided to choose the safe, well documented and widely used bacterial E. coli DH5a strain and E.coli BL21 (DE3) strain, which are on the White List provided by iGEM Competition. These E.coli bacterial strains are classified as Risk Group 1 microorganisms according to the current edition of Microbiological and Biomedical Laboratories (BMBL) and Deutsche Sammlung von Mikroorganismen und Zellkulturen / German Collection of Microorganisms and Cell Cultures (DSMZ). Risk Group 1 microorganisms are unlikely to cause harm to the environment or disease in healthy adult humans. This statement can be found in [WHO's Laboratory Biosafety Manual and European Union Directive 2000/54/EC] for the protection of workers from exposure to biological agents.
Human cell lines information
Human cell lines that have been tested and certified free of known pathogens, according to iGEM White List were also utilized. More specifically A549 and MRC5 cell lines were used for further validation of our detection system's performance. Both cell lines can be used in a BSL-1 laboratory according to American Type Culture Collection (ATCC) [product sheets] and the Microbiological and Biomedical Laboratories (BMBL).
Our project's goal is the construction of the detection device DIAS for the quantitative measurement of miRNA biomarkers in patients' blood samples. For this purpose, we exploit the CRISPR/Cas13a system combined with microfluidic technology. Considering the safety aspects of this project, the diagnostic procedure does not endanger the patient since it only requires a blood sample. This examination can take place only in appropriate centers such as a hospital or a diagnostic center. Thus, the patients' examination will be conducted by qualified staff for their thorough monitoring in appropriate conditions. During our project development and before the iGEM competition we filed a provisional patent in the USA. Our aim is the construction of the detection device DIAS and its commercial availability in accordance with the requirements of ISO 14971:2019 Medical devices — Application of risk management to medical devices.
In conclusion, our system's functionality does not require the presence of bacteria or any other microorganism, thus it is absolutely secure for humans and the environment. All parts and constructs utilized or designed during our project's implementation are on the iGEM White List and before their lab use, we verified and clarified the absence of any risk to humanity and the environment. Generally, our project is totally in line with all safety requirements given by the iGEM competition.