As per the Macquarie University Biosafety Committee (IBC) and workplace health & safety guidelines, we were required to complete a laboratory safety induction. This induction was performed by the head laboratory technician and involved the demonstration of correct safety protocols such as the safe handling and use of the autoclaves and microwave and their contents as well as necessary emergency evacuation and power shut off procedures and all relevant chemical safety data sheets of all chemicals used. Additionally we were shown the locations and usage instructions of the fire and chemical extinguishers; fire blankets; emergency showers and eyewash stations. Correct laboratory attire has been consistently enforced in lab coats, safety glasses and nitrile gloves, as well as closed toed shoes and appropriately tied back long hair.
An advisor or laboratory technician was always present within the laboratory available for consultation. Prior to using any equipment we were briefed on the safe usage procedures. All solvent extractions were performed in a fume hood to eliminate risk of inhalation and biological hazard usage and disposal procedures were adhered to by maintaining aseptic by sterilizing the workspace with ethanol before and after use and disposal of contaminated waste into designated biohazard waste material bins to ensure no accidental release of our modified strains into the environment.
The two strains of Escherichia coli used were DH5-Alpha and NEB® Turbo Competent (High Efficiency) are both derived from the K12 strain are classified as exempt dealing GMO under the Gene Technology Act 2000 by the Office of the Gene Technology Regulator (OGTR), the main regulatory body of Australian gene technologies. These strains are non-pathogenic and can be handled at a biosafety level of 1 and present little hazard risk to laboratory personnel as it is not known to consistently cause disease in healthy adults. The strains include mutations in the recA pathway which reduces homologous recombination and do not readily produce thiamine and leucine, as a result they cannot survive unless provided with a source of these amino acids.
The chlorophyll biosynthesis genes used in our product originated from Chlamydomonas reinhardtii are not readily found within E.coli or any other variants of these genes. The genes would not give the modified organism any survival advantage or resistances besides the allowed chloramphenicol resistance gene found on the plasmid backbone supplied by iGEM. The protein expression of the plasmid is inducible with IPTG or similar analogues and is inactive otherwise, giving greater control over when the chlorophyll biosynthesis genes are being produced. The presence of chlorophyll, its precursors and the enzymes involved with its synthesis within the organism is unlikely to be advantageous as it involves the production of many nonessential proteins and the rerouting of essential biomolecules for example protoporphyrin IX away from its associated metabolic pathways. In addition the presence of the chlorophyll and the associated vesicles is likely to lead to an increase in reactive oxygen species from exposure to sunlight as well as a crowded cytoplasm. For these reasons it is unlikely the plasmid would offer any advantage to the survival of E.coli cells and is only viable in a controlled laboratory setting.
Australian Government, Department of Health – Office of the Gene Technology Regulator. Gene Technology Act 2000. (Commonwealth).