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Safety forms were approved on September 22, 2013 by Evan Appleton.

Experimental safety

Our research is done in a GMO class 2 laboratory at the faculty of Bioscience Engineering at Ghent University. Our bacteria, Escherichia coli strain DH5a, Escherichia coli Top10 and Escherichia coli K-12 MG1655 are standard non-pathogenic laboratory bacteria belonging to risk group 1. According to this classification it is unlikely that they will cause human or animal disease. For the P1vir phage transduction extra safety measurements to prevent contamination in the lab were taken. Phages are viruses that infect bacteria only, they do not infect humans, animals or plants and are therefore officially allocated to the risk group 1. The P1vir phage we used is a virulent mutant of phage P1 used for transduction experiments.

Every person who works in one of the labs of Ghent University must know and act according to certain regulations. This means that each team member signed the Laboratory and workplace regulation in which he/she declares to have taken note of these regulations, to have received a specimen of these regulations and to follow to the guidelines/obligations who arise out of these regulations. This also includes always wearing a lab coat and gloves when necessary. In addition every one of our team got a safety and waste disposal training before performing our experiments in the lab. This training focused especially on the safe use of ethidium bromide, which we use as dye for DNA detection in gels. During the eventual execution of our experiments, we were supervised by our instructors who made sure we performed everything according to the safety regulations. In case of phage contamination, there are two controllers in our lab who take care of it. They encourage all employees and students to do as much as possible to prevent phage contamination. In our lab, all glass work that has been in contact with biological material is disinfected with bleach and autoclaved before it is re-used, making sure no living micro-organisms are remaining. When applying bleach, we wear gloves as it can cause skin irritation. Also, benches and flows are regularly cleaned with Umonium, a broad activity spectrum disinfectant. Additionally any cultures that leave the lab are tested, in order to prevent contamination of other external laboratories. In case of suspected contamination, an assay to determine very low titers of bacteriophages in solutions is used before further use.

All experiments were carried out in a contained laboratory environment (GMO-class 2 laboratory). If containment fails, however, our project poses minimal risks for public and for the environment. Bacteria often have the capability of transferring genetic material, especially when self-transmissible vectors are used. As often done in practice, the vectors in our experiments are not self transducible, avoiding genetic material from being easily transferred.

The basic idea of our project is the augmentation of the environmental safety of the already existing and very useful CIChE technique (Chemically Inducible Chromosomal Evolution), which in its original form incorporates many copies of an antibiotic resistance gene in the E. coli genome. Even though the DNA cassette’s homologous regions have low affinity to the genome of E. coli (source: Tyo et al., 2009) and the genome of other (possibly pathogenic) strains, horizontal gene transfer cannot be entirely ruled out. If horizontal gene transfer, however very unlikely, would take place with the attained bacteria of our improved technique, the selectable marker transferred would be the ccdA gene encoding the antitoxin CcdA, instead of the antibiotic resistance gene. This way, using the CIChE technique no longer implies a risk of contributing to the current issues with spreading antibiotic resistance.

Biobrick safety

Our new BioBrick part is based on an already existing one, in which we inserted a T7 promoter in front of the already present ccdB gene. This modification does not raise any safety issues, as no possible hazardous products are formed.

Safety Committee

The Faculty of Bioscience Engineering at Ghent University, where our lab is situated, has its own facultary Biosafety Committee (only Dutch version of the website available). This committee makes sure the researchers are aware of the European and Belgian legal framework concerning biosafety. All activities of this committee take place in close consultation with, amongst others, the facultary Environment, Hygiene and Safety Committee and with some Rectoral Services.

Ghent University has its own Laboratory and workplace regulation. Guidelines more specific for working with GMOs can be found in the Biosafety in the laboratory document provided by the Flanders Interuniversity Institute for Biotechnology.