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Fig. 2 | BMC Research Notes

Fig. 2

From: Quantifying heterologous gene expression during ectopic MazF production in Escherichia coli

Fig. 2

Increase in the mean level and variation in fluorescence during mazF overexpression. A The increase in fluorescence of bacterial cultures was determined by comparing two flow cytometry time points, measured 2 and 6 h after inducing mazF expression. The fluorescent gene reporters were encoded on a high-copy (HC) or a low-copy plasmid (LC), transcribed to a leaderless (without 5′-UTR) or a canonical (containing 5′-UTR) mRNA that contained ACA sites (mCherry) or was devoid of ACA sites (gfpΔACA). The highest increase in fluorescence was detected from the can-gfpΔACA reporter encoded on a high-copy plasmid, which was almost twofold higher than the fluorescence increase measured from the ll-gfpΔACA reporter encoded on a high-copy plasmid (N = 3 independent replicate cultures for GFP fluorescence analysis, N = 12 for mCherry fluorescence analysis). B Coefficient of variation (CV) in mCherry fluorescence was calculated as standard deviation divided by the mean of the log10-transformed fluorescence data, for different phases of bacterial growth, and it is a proxy for population heterogeneity [9]. CV in mCherry fluorescence increased by 32.4 ± 19.4% in mazF-induced cultures, during 4 h of mazF overexpression (N = 12 independent replicate cultures, p-value = 0.0001). C Green distributions depict measurements of the E. coli strain BW27784 harboring the plasmid pBAD-mazF and the can-gfpΔACA reporter encoded on a low-copy plasmid. Light grey distributions depict measurements of the strain harboring only the plasmid pBAD-mazF. 0.02% Ara was added to exponentially growing cultures to induce mazF overexpression, and flow cytometry analysis was performed in the early exponential phase, and 22 h after mazF overexpression [OD600(uninduced) = 5.31, OD600(mazF-induced) = 3.20]. After 22 h, mazF-induced cultures were comprised of bacterial subpopulations of different GFP fluorescence intensities, while uninduced cultures exhibited unimodal distributions of GFP fluorescence

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