Generation of a bacterial clone for assessing the impact of climatic stress conditions on microbial proliferation

Documented increases in atmospheric Carbon Dioxide (CO2) concentrations have contributed to a rise in average global temperatures. Environmental variation due to climate change is expected to affect the growth of microorganisms. Hence, there is a need to assess the induced adaptations of microorganisms, which are common biological contaminants, to environmental changes. Therefore, an enhanced green fluorescent protein (eGFP) expressing Escherichia coli BL21(DE3) clone was generated. Plasmid pAP1698-4 was used as the donor for the eGFP gene and pD454-MBP as the recipient plasmid to produce pD454-MBPeGFP. Expression of eGFP in the clone was confirmed using confocal microscopy. The growth of the clone was characterised by plate counting technique. Variation in the length of the lag phase, λ, and growth rate, μmax, kinetic parameters of the clone was observed, compared to the wildtype BL21(DE3). A live/dead kinetic assay, using eGFP for the quantification of live cells and propidium iodide (PI) as a stain for dead cells, was optimised using a microplate reader with controlled temperature and CO2 conditions. Full growth curves were collected when culture media was inoculated with 4 to 6 Log10CFU.mL-1. The optimal PI concentration was 150 nM; higher concentrations inhibited growth, and lower concentrations gave no signal difference compared to the blank. The growth kinetics of the clone under different environmental conditions; between 400 ppm to 2500 ppm CO2, combined with 37°C to 42°C, were evaluated using the live/dead kinetic assay, allowing assessment of response to induced environmental stress.