Lab Safety
Throughout the project, an advisor or lab technician was always present in the laboratory for consultation and supervision. Prior to the use of any equipment, a briefing on proper procedures for safe operation was given.
Personal protective equipment was used, such as wearing lab coats, safety glasses, nitrile gloves, enclosed-toed shoes, and tying back long or loose hair appropriately.
Strict usage and disposal procedures for biological hazards were followed, maintaining aseptic workspaces and disposing of contaminated waste in designated biohazard waste material bins. These procedures ensured that there were no accidental releases of our modified strains into the environment.
Lab Environments
Project Safety
Accidental Releasae
The strains of E.coli that were used were all derivatives of K12 and require thymine for growth in an external environment. Our strains cannot independently produce amino acids or vitamin B5, preventing them from surviving in variable environments. Furthermore, K12 strains contain mutations that suppress pathogenic qualities of E.coli. These strains, employed as part of genetic engineering methodologies in an educational environment, are classified as exempt dealing GMO under the Gene Technology Act 2000 by the Office of the Gene Technology Regulator (OGTR) thus, free from regulation. This category includes K12, B, C, and Nissl strains.
Additionally, due to the rec-A minus gene of these strains, they do not possess the ability to engage in homologous recombination, limiting the spread of genes amongst other bacteria strains.
Further, the T7 promoter used for controlling Green Fluorescent Protein (GFP) fluorescence is a B strain. As such, it is considered safe for use in our applications.
Kill Switch
Upon accidental release, exposure to standard atmospheric conditions of oxygen levels of ~21%, the FNR would deactivate the nitrogenase cycle. Despite having been designed to optimise the bacteria to ideal oxygen concentrations of ~2%, the system can double as a kill switch to leakages, as the FNR promoter limits production beyond ~18% oxygen, with a GFP intensity per OD600 approaching close to 0 past 18% as displayed in Fig 2.1.
Citations
Australian Government, Department of Health – Office of the Gene Technology Regulator. Gene Technology Act 2000. (Commonwealth).
Chen, Z., Yang, H., & Pavletich, N. P. (n.d.). Mechanism of homologous recombination from the reca–ssdna/dsdna structures. Nature News. https://www.nature.com/articles/nature06971
EA;, A. B. (n.d.). Complete genome sequence of NEB 5-alpha, a derivative of escherichia coli K-12 dh5α. Genome announcements. https://pubmed.ncbi.nlm.nih.gov/27834703/
