Generating a bacterial clone for evaluating growth under stress

Abstract

Climate change is a major global concern, with implications regarding (amongst others) changes in the universal microbial proliferation due to alterations of the environmental conditions. Subsequent bacterial adaptation to climate variation (i.e. acclimatisation) can result in an effect, as of yet unknown, with regards to their ability to grow and propagate. The project aimed to create an Isopropyl β- d-1-thiogalactopyranoside-inducible enhanced Green Fluorescent Protein (eGFP) synthesising reporter Escherichia coli clone via chemical transformation, which would then be used as part of an eGFP/Propidium iodide (PI) cell viability assay. Information obtained from this model organism should be applicable to other Gram-negative organisms and allow for a complete understanding of the mechanisms of bacterial response to environmental stress. The study generated E. coli BL21 (DE3) pD454- MBPeGFP, validated and characterised it. A 150 nM PI concentration to be optimal for the eGFP/PI assay, however the use of PI was determined to be inappropriate to kinetic assays. Growth was assessed using three parameters: lag (λ), maximum growth rate (µmax), and fluorescent time to detection values (FTTD). λ decreased with increased carbon dioxide (CO2), and temperature. Values for µmax were not affected by increases in CO2 but diminished with increased temperatures. FTTD values demonstrated its application to assessing changes at lower levels of inoculum; application may vary according to a study’s objectives. Future work should aim for characterising responses to changes in other variables such as pH.