Introduction
To ensure the safety of our iGEM project team and prevent incidents, we established a safe working environment by implementing various laboratory safety measures. The primary goal of safety and security is to minimise the risk of harm in all areas, including irresponsible conduct, accidental exposure, environmental release, and intentional misuse. We strictly adhered to the safety guidelines set by the iGEM committee throughout the project. This included assessing and minimising all potential risks associated with our wet lab procedures before starting, such as working with genetically modified organisms, hazardous chemicals, and equipment.
Avoiding irresponsible conduct
The project included work with Biosafety level 1 (BSL1) approved organisms (E. coli DH5-alpha and Saccharomyces cerevisiae CENPK113-3C). The safety of these strains has been well-documented. Nevertheless S. cerevisiae is a potentially spore-forming fungus and required check-in approval from the iGEM Biosafety Committee. We did not test our biosensor on humans. Our experiments did not involve animals or animal samples. Gene drives are not included in our project. We did not use novel resistance factors, add known factors into organisms that were not used before or use resistance factors not previously used in our facility. We did not obtain any parts or organisms from non-traditional suppliers or from the environment. In our project, we used environmental samples that could contain microorganisms, which pose a risk. However, we did not use any microorganisms or their parts from these samples in further experiments.
Before starting our wet lab experiments at the university, we completed a compulsory safety and risk assessment for our experimental work, as required by Danish law regarding the Lab safety. The main goal of this assessment is to establish the safest working methods and ensure a safe working environment. We reviewed various safety documents provided by DTU to help identify potential project-related risks and their mitigation strategies in line with Danish regulations on handling GMOs. We then had to pass a detailed quiz based on this information to gain access to the DTU laboratories. Additionally, it was mandatory to participate in a lab safety tour led by a laboratory technician. We received thorough safety training on how to deal with physical, biological and chemical hazards in the genetic engineering laboratory and the chemistry laboratory, use of biosafety cabinet, chemical fume hood, personal protective equipment, accident reporting, good microbiological technique, autoclave use for sterilisation and about emergency procedures. This tour provided us with practical knowledge of emergency procedures and lab policies. Furthermore, before using any potentially hazardous equipment in our labs, we had to undergo mandatory training sessions conducted by our laboratory technicians.
Preventing accidental exposure
Work involving BSL1 organisms was entirely carried out in permitted Genetic Engineering Class 1 Laboratories and following the laboratory regulations.
Incorrect handling of carcinogenic and toxic materials could result in the exposure and negative health impact of the users and third parties. Therefore, all of the material handlings are to be performed with adequate personal protective equipment. Every member was equipped with protective equipment consisting of a lab coat and safety goggles. Disposable gloves were available at every working station.
We used some chemicals that are hazardous, such as SYBR Safe (safer alternative to ethidium bromide) and hormones. SYBR Safe is a mildly mutagenic substance used for visualising DNA during gel electrophoresis. It is considered a safer alternative to ethidium bromide, as it induces fewer mutations. However, aside from its mutagenic potential, SYBR Safe can also irritate the skin and eyes and is suspected of contributing to organ damage. We run all the gel electrophoresis in the chemical fume hood to avoid contact with the SYBR Safe DNA Gel Stain. We decided to modify our handling of the human hormones because of the carcinogenic risks. Our supervisors dissolved and diluted the hormones for us to work with because of the higher risk when working with the powdered forms. The final working solution of hormones was 784 mM or less for all hormones after dilution. Along with these precautions, we ensured adherence to special instructions given by the official DTU website, Kemibrug, for identification of chemicals before use.
In case of an accidents/emergency the procedure is to stop the accident, create safety, assess the situation/the injured, call for help - call 112 (the number for the emergency services in Denmark), provide first aid or other appropriate help, meet and guide the emergency services, inform the DTU corporate emergency team. We also always work with at least two people in the laboratory.
All accidents or near accidents (both severe and minor), must be reported and documented. In this case, we inform a technician, our supervisor and the DTU Safety Group of accidents. The Safety Group is responsible for filling an official report of the accident to be used for the improvement of the work environment and in case of subsequent insurance claims by us.
Mitigating environmental release
Adequate waste management and laboratory infrastructure prevents the escape of biological substances or organisms outside the laboratory and proper disposal of hazardous waste. We were autoclaving all biological waste in the GMO level 1 laboratory. In case of eventual spills we followed the following procedures. Powders spilled while weighing should be cleaned up immediately using water or other solubilizing solvents and tissue paper. Liquids spilled on the floor should be mopped up with tissue paper or cat litter, and disposed of based on the nature of the liquid. Decontaminate microbial spill using 70% ethanol and tissue paper.
We did not work in conditions where sporulation of S. cerevisiae has been described, as we worked with a haploid strain, and only starved diploid yeast can sporulate. We worked in conditions that did not cause our strain to become diploid (30°C and pH = 6).
The final use of the cell-free biosensor is thought to take place outside of the laboratory. The use would include a small enclosed device into which a water sample would be loaded followed by a detection of a signal directly from the device. However, due to time limitations, this was not the scope of the iGEM project and our device or any biological material were not used outside a laboratory.
Risk Assessment and Expert Feedback on Sensor Safety
Due to the nature of our project the risk of intentional misuse is low, we thought of possible scenarios but our sensor does not provide numerical values nor is it tailored for specific compounds and cannot therefore be utilised by people that want to synthesise potentially hazardous compounds (for quality control).
When it comes to environmental safety the lack of protein coding genes and the intended use setting, lower the risk. We are here referring only to the biosensor and not the human hormone receptor producing yeast cells or the in-cell assay, as none of those will be present in the final product. In the sensor there is no protein coding sequence that can be passed via horizontal gene transfer to any organism in the wild, although we should note that the broccoli aptamer is an artificial gene, not found in nature. After receiving advice through our human practices we decided that our sensor should not be available for domestic use and will be used only by professionals. That is not strictly limited to an environmental setting, but it reduces the total number of units used and the probability of their improper disposal.
Last but not least we should comment on the number of experts we contacted through our human practices and their take on safety. Many of these people have years of experience in fields related to safety, whether they are researchers in Academia (Dr. Gerd Seibold, Dr. Mogens Kilstrup, Dr. Terje Svingen, Dr. Brigit van Brenk), doctors (Svend Lindeberg), politicians (Magnus Heunicke, Franciska Rosenkilde, Sofia Osmani) or experts, product managers, directors holding responsibilities in companies and institutions (Ida Holm Olesen, Susan Münster ,Niels Bjerre, Magnus Löfstedt, Thomas Hansen, Christian Ege to name a few). These people run companies, environmental agencies, and institutions charged with our safety. When visiting the Lyngby waterworks we were explained that they are concerned with cyber attacks and terrorism with the related infrastructures being prime targets and the infrastructure. Yet none of them raised concerns about possible misuse or environmental implications of our sensor. That didn’t affect our risk analysis activities nor eased our safety concerns but it is a good indication. The only relevant comment on any risk was on the psychological effect it can have on people (panic), something addressed by our team with a radical redesign of our targets.
