Safety
Project 
SAFETY
Working in a laboratory environment and especially working with GMO’s comes with a lot of responsibilities and safety considerations. The labs we are using for our iGEM project experiments are the same labs we use for most of our practical courses and research projects in the Maastricht Science Programme of Maastricht University. Our building is located on the Brightlands Chemelot Campus in Geleen, the Netherlands, a research and development (R&D) campus, mainly focusing on chemistry and material sciences, that houses several small companies and research infrastructure. In addition, there are several chemical plants and storage facilities for hazardous chemicals. That means that access to the campus is restricted and we are only allowed entrance after receiving the campus safety and access instructions.
Our university and study program also immensely emphasizes safe working practices in the lab environment. At the beginning of our studies, we all receive a guided tour around the campus and a thorough safety lecture. This lecture deals with the campus and building specific safety issues like evacuation plans, different siren alarms, and locations of RDR (toxic shelter) rooms. Additionally, it covers the emergency equipment like safety showers, eye wash stations, and first aid materials as well as general lab safety rules, like the use of specific protective clothing, how to handle chemicals, the use of safety data sheets, chemical and biological waste disposal, amongst other key topics.
For working with GMOs, some additional rules apply. Both of our supervisors have years of experience in working with organisms, GMOs in general and with the organisms used in our project. Our Principal Investigator (PI) is also a departmental biosafety expert and the responsible investigator (RI) for our biosafety level 1 (ML-1) lab, thus he gave us additional work and lab instructions. We also received additional safety information through an instructional video from the ‘National Institute for Public Health and the Environment’ (RIVM), an independent agency of the Dutch Ministry of Health, Welfare and Sport.
Getting lab specific instructions minimizes the risk of contamination and the escape of microorganisms into the environment. These instructions involved the use of PPE (such as dedicated lab coats and gloves for working with microorganisms) and regular sterilization of equipment and benches. All GMO related experimental work is performed in the designated ML-1 lab.
All chemicals in the experiments, such as dyes and solvents, that are considered toxic were only used in a fume hood while wearing protective equipment like gloves and goggles. All material and small lab equipment used in the experiments were first sterilized. Possible biologically contaminated material and waste products were inactivated using an industrial autoclave. The accuracy of the autoclave is regularly tested to ensure a proper inactivation of biological material.
The procedures and guidelines depend on the restriction level of the GMO activities, decided by the Dutch Ministry of Housing, Spatial planning and Environment (VROM).
Activities done at Maastricht University that involve GMOs and potential pathogenic microorganisms fall under several rules. Genetically Modified Organisms fall under the GMO decision and regulation. Implementation and execution of this decision are mandated to the directors of the administrative units and to the Biosafety Officer, by the Executive Board (Maastricht University) and by the board of the Maastricht UMC+ (University Medical Center). Our PI is in contact with the university's Biosafety Officers and registers employees working under his responsibility for approval as GMO workers. All MSP-Maastricht iGEM wet lab team members were approved by a Biosafety Officer to work with GMOs.
All organisms and constructs that are used in the ML-I lab are evaluated using a risk assessment tool. Using this tool, the correct restriction level can be determined and evaluated based on the host organism, vectors, donor sequences and activities.
For the development of our siRNA based biological pesticide against the oak processionary caterpillar, in procedures such as cloning, DNA extractions and assemblies, we only used standard lab risk group 1 microorganisms like E. coli DH5α strain.
For the final application, the expression of the siRNA, the risk group 1 bacterial E. coli strain HT115(DE3) is used as the chassis. This strain is an RNase III-deficient strain with IPTG-inducible T7 polymerase activity and tetracycline resistance.
Some of the parts including promoters, siRNA sequences, and terminators used in the project are new but were subject to an internal risk assessment required for national and the university safety regulations as mentioned above. All constructs are considered level 1.
DNA was extracted from the oak processionary caterpillars (Thaumetopoea processionea) and used for PCR. The sequences of the siRNA are based on essential genes with unique sequences from T. processionea; meaning that they are highly specific for the target organism by design. The siRNAs will be expressed in E. coli which will then be fed to the caterpillars. The siRNA sequences for the target genes that we ultimately use are (by design) safe to the surrounding environment and humans since they only target the genes of these caterpillars.
As the OPC's live on oak trees, the initial idea is to spray our developed and engineered bacteria expressing the siRNA on the trees, similar to how it is done nowadays to fight the pest by spraying the trees with Bt toxins (Bacillus thuringiensis).
The possible risks of using the caterpillars in the experiments is reduced by only working with them in the developmental stages where they do not produce the potentially harmful hairs (instar stages 1-3).
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