Safety in the laboratory

Working with cancer cell line Caco-2

Cell cultures of cancer lines are very sensitive in their management, and they should be treated in an utterly safe and sterilized environment. Our university has great experience in handling cell lines and provides specific spaces for working with them. Those spaces are equipped with well-maintained and up-to-date flow hoods with specific lab equipment and tools (e.g. pipettes, tubes, gloves, vortex, incubators, etc.) that are specifically only for use for experiments that use cell lines.

Firstly before getting in hand with our experiments, our graduate supervisor trained us to handle the cell lines and maintain every safety requirement needed.

Safety in the laboratory

Working on an open bench

While working with non-hazardous or non-sensitive substances, we still had to maintain the safety rules on the open lab bench. We had our eppendorfs and other containers sealed very well, while the tips of the pipette were thoroughly changed. The boxes with the new tips were always autoclaved, and the lab bench after every experiment was cleaned with ethanol.

Safety in the laboratory

Working with the ELISA kit

ELISA is a very long and detailed experimental procedure that is performed on an open bench but still, safety needs must be followed. The kit included specific orders that we followed regarding corrosive materials that were included in the reagents. Every member of the wet lab wore gloves and a lab coat and discarded the remaining corrosive substance in a special biohazard container intended for biohazardous substances.

Safety in the laboratory

Working with microorganisms

Microorganisms specifically bacteria need specific conditions for growing and culturing. In the Laboratory of Biotechnology and Applied Microbiology which is specifically designed for culturing microorganisms and conducting research on various food sources, there is specific equipment for that kind of lab work. There are multiple autoclaves, flow hoods, incubators, and technological equipment such as HPLC (High-Performance Liquid Chromatography). The laboratory personnel is highly equipped to handle those pieces of machinery as well as culturing microorganisms and experimenting with food substances. Thus, all the safety measures were followed. Lastly, since the Laboratory of Biotechnology and Applied Microbiology is included in the FoodBiomes, a Hellenic distributed research infrastructure, all the equipment used is certified.

Safety in the laboratory

Personal safety

All our members of the wet lab team wore their lab coats and gloves as it was necessary for specific procedures that required sterile conditions and protection from corrosive substances.

Biological Safety

Strain selection

Besides the scientific and bioinformatic research that was conducted in order to choose our strain, we also wanted to ensure the safe aspect of the strain that could be potentially administered to a patient. Lactobacillus rhamnosus GG is very well-known to consumers and widely used as a probiotic since it is Generally Recognized As Safe (GRAS) by the Food and Drugs Administration (FDA) for its administration and addition in foods.

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Biological Safety

Generally GMOs as food substances

According to the FDA and the Qualified Presumption of Safety (QPS) self-cloning, which is the reintroduction of the DNA from a host that modifications have been done or from a closely related organism that is stated as GRAS, is not considered a genetically modified organism. Therefore, under that umbrella the organism that we aim to introduce as a probiotic is recognized from a wider perspective safe.

Biological Safety

Plasmid design

The plasmid that we designed to be introduced into our strain Lactobacillus rhamnosus GG, and that will include the genes for the butyrate-producing metabolic pathway should meet certain safety requirements:

• The DNA of the plasmid should come from the same organism or from an organism that is recognized as safe and is closely related to the host. This reassures that the bacterial organism pruned to be a probiotic, is safe for the human being, and will not be the cause of undesirable side-effects and conditions that jeopardize the overall health.
• The plasmid must not include an antibiotic resistance gene as a selection marker. In this way, the “cross-over” of antibiotic resistance genes between microorganisms in the gut flora and therefore any chance of an antibiotic resistance episode occurring is not viable.

If the above requirements are met, then we have created a food-grade system. The design of our plasmid is based on the above safety pillars. The pMG36e plasmid is a vector that is derived from Lactococcus lactis a GRAS organism and very closely related to the Lactobacillus species that is our target host.

Unfortunately, the few commercially available Pmg36e plasmids contain antibiotic resistance to the ampicillin gene. However, through cloning, we can add another marker that bibliographically is very used and safe, the NisI gene from the NICE operon that codes for an antibacterial peptide that grants immunity from nisin which serves as a food preservative.