There is a famous quote that goes: “Safety doesn’t happen by accident”. Indeed, safety is an important factor in research that must be considered carefully. Therefore, safety is a value that comes back in all aspects of our project. Find out how we integrated it in each of the aspects below.
General lab safety
We are very lucky to have had our very own lab space during iGEM; a cozy ML-1 lab in the new Applied Sciences building of the TU Delft campus. ML-1 is the lowest safety level when working with genetically engineered microorganisms, meaning that experiments involve low or no risks (e.g. no human pathogens). This level was sufficient for almost all of our experiments, which consisted predominantly of cloning (i.e. Gibson assemblies and restriction-ligation), transformations and PCRs.
Before we started with this work in the lab, all team members had to pass different safety tests and receive training during an introductory wetlab week. Here you can think of an obligatory ML-1 safety training and test, a chemical safety training and test and a general safety training and test (with regards to contact persons, escape routes and meeting points). During this week we were instructed on how to work safely in ‘Ethidium-Bromide’-zones, i.e. lab spaces that were possibly contaminated with compounds used for staining DNA and proteins.
Besides these trainings, safety was strived for by thinking the experiments through beforehand. For this purpose, our team Safety Manager, Isabell Trinh, wrote safety proposals for our experiments. These proposals were set up in collaboration with and approved by Susanne Hage and Marinka Almering. Susanne Hage is the wetlab coordinator of the department of Bionanoscience of the TU Delft and Marinka Almering is the Biosafety officer of the Faculty of Applied Sciences of the TU Delft. These proposals also contain safety precautions and disposal requirements that can be found here.
Other than the molecular work, we also purified and characterized proteins and vesicles. For these experiments we often required more specialized equipment that was not present in our own lab. In these cases, we could use the equipment of the Department of Bionanoscience of the university. We also received explanation/training before we used any of this equipment on our own. For example, all team members completed a basic laser/microscopy safety training.
All our research adheres to the biotechnology regulations of biosafety for The Netherlands.
Our ML-1 Lab
Safety of our project
Safety considering ML-1 and ML-2 level
Besides the general safety that is required to responsibly work in the lab, there were also aspects more specific to our project. Firstly, we worked with the host organism Escherichia coli. The wildtype of this strain is typically a harmless lab pet of Risk Group 1. We performed several transformations for our experiments, using different derivatives of this wildtype and conferring new properties to these bacteria. In general, the E. coli K-12 strain TOP10 was used for cloning and the E. coli BL21, BL21(DE3) and Dh5α strains were used for protein expression after correct cloning. Furthermore, the vesicles module often used a special hypervesiculation strain in their experiments, which is called KEIO.
If genetically engineered bacteria, like the ones we worked with, are put in a natural environment, the outcome is very unpredictable. The microorganism could disrupt the balance that evolved in this environment, drastically changing it. Therefore, we strictly adhered to the ML-1 safety rules. Autoclaving our waste and washing our hands before leaving the laboratory are just two examples of how we ensured that the microorganisms were contained in the lab. Other measures are sterilizing our work space with 70% ethanol before and after working with microorganisms. Furthermore, we ensured that less experienced team members were always accompanied by team members that had more experience.
However, the ML-1 level did not suffice for all our experiments. To prove that our applied design indeed functions properly, we needed to detect antibiotic resistance genes of which we did not know the exact sequence. Furthermore, we worked with an actual milk sample from a farmer and of course the exact (microbial) contents of this sample were unknown to us. Therefore, the ML-2 biosafety level was required for some parts of our project. For this purpose, one of our team members, experienced with the ML-1 level, was trained to work in the ML-2 lab of our supervisors. Even after the training, she only worked in this lab under supervision.
Safety considering chemicals and methods
Besides microorganisms, we also handled various chemical compounds with which we should be careful. For example, we worked with SYBR Safe for the staining of DNA. Harmful substances such as this were always handled responsibly, i.e. while wearing gloves and (where applicable) other protective clothing. Here, we took care that the ‘clean’ environment was not contaminated with the harmful compounds we were handling by only wearing one glove (to open doors etc. with the ‘clean’ hand). However, in designated ‘ethidium-bromide’ areas, we made sure to wear two gloves to protect ourselves against contamination. Furthermore, when we excised bands from an agarose gel, which happens under illumination by UV-light, gloves and an UV protective screen were used. Finally, a common safety measure was to work in the fume hood instead of on the open bench. This was for example the case when we prepared antibiotic stock solutions.
Safety considerations in the applied design
Early on, we set out to make our project safe for on-site usage, which in this case means on a farm. The current (European) ML-1 safety regulations do not always allow the use of genetically engineered organisms outside the contained lab environment. Furthermore, through stakeholder interactions (Integrated Human Practices), it soon became clear that it would be best if our final product would be completely cell-free. Therefore, our tool was always designed to only contain purified proteins and no bacteria. In our collaboration with the RIVM (The National Institute for Public Health and the Environment) we even went one step further and tried to educate other engineering teams on how to incorporate safety and responsibility in their design through a Video Project.
Our project might be believed to be too ambitious, considering a farmer now must determine himself whether his sick cow is infected with antibiotic resistant bacteria. Nevertheless, through continuously designing, evaluating and redesigning our sample preparation, we have come to a protocol that is easy to follow and that eliminates possible risks. To give an example, one of the first steps in the protocol is the boiling of the bacteria. This makes our protocol safe in two ways: (1) no microorganisms will survive this step, so the farmer does not have to continue with a sample containing living bacteria; and (2) the boiling step is safer than the alternative microwaving of the sample. Although the latter method was previously preferred due to its simplicity, we experienced problems with this method in the lab. Therefore, we chose safety over simplicity in this step and changed it to the currently incorporated boiling step. Moreover, all equipment required can be bought in the supermarket and is thus user-friendly. Our final measure to make the design safe, is that we indicate clearly that some parts of the protocol should not be carried out by the farmer himself, but by specialists. This way we prevent a lack of knowledge on certain techniques to pose a risk to the safety of the farmer and his environment.