Investigating Flow Cytometry as a Potential Method for Real-time Analysis of Gene Expression following Escherichia coli Transformation

Bacterial transformation is commonly detected by growing colonies on selective media. This process is time consuming and labour intensive. The aim of this study was to develop a more efficient real-time method to observe gene expression following transformation of Escherichia coli with plasmid DNA. In this study, we investigated flow cytometry as a potential method for studying transformation. Rather than relying on colony formation to detect transformation, we hypothesised that flow cytometry could be used to detect transformation of single cells shortly after plasmid update. We used pGLO, a plasmid which carries a gene coding for green fluorescence protein gene linked to an arabinose inducible promoter.  pGLO was used to transform BL21 E. coli. Fluorescence microscopy was used to observe cell morphology and to observe GFP expression over time following induction with arabinose. E. coli cells showed morphological changes during increased incubation times. The transformed cells showed green fluorescence 40 minutes after induction with L-arabinose.  Using flow cytometry, we were able to detect cells expressing green fluorescence in cultures diluted 10^-4 through to 10^-7, which are expected to have 4000 cells/ml to 4 cells/ml respectively. We were able to detect 3 GFP positive cells in 3180 total cells using flow cytometry. Most notably, newly transformed cells were detected by flow cytometry 1 hour post-transformation. Taken together, these data suggest that flow cytometry can be used to study plasmid transformation and gene expression in real-time.  It is also worth noting that variable cell shapes may interfere with measurements; however, this issue may be circumvented by reducing growth time after transformation, with flow cytometry analysis being completed within one hour of transformation.