Fig. 4: The lacI non-canonical GTG start codon fosters lactose utilization in vitro.
a, Effect of the start codon sequence on the LacI expression level. Soluble extracts of E. coli 8178 strains (WT, ATGlacI, GTGlacI and lacI mutant) were subjected to a 12.5% acrylamide sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis followed by an immunodetection step using an antibody specific against LacI (upper gel) or the subunit-β of the RNA polymerase (lower gel). The detected LacI protein is indicated on the right. The molecular weight marker (in kDa) is added on the left. The western blot was performed independently twice and a representative experiment is shown. b,c, In vitro growth assays. The indicated strains were individually grown on a minimal medium supplemented with lactose (10 mM). Bacterial growth was quantified by measuring the optical density (OD) at 600 nm (OD600). Strains were previously incubated overnight in a minimal medium supplemented with glucose (20 mM) (b) or in a rich LB medium (c). The solid coloured lines represent the means of biological triplicates, and the error bars represent standard deviation. d, The lacZ expression level is influenced by the lacI start codon sequence. The lacI, WT, ATGlacI and GTGlacI strains were transformed with the p-PlacZ-gfp reporter plasmid. The strains were incubated in a minimal medium supplemented with glycerol (50 mM) in the presence of the indicated IPTG concentrations. The gfp signal resulting from lacZ expression at mid-exponential phase represents the mean of a biological triplicate and is expressed as the mean fluorescence intensity (MFI) normalized by the bacterial density (optical density unit: uOD600). The error bars represent standard deviation. Student t-tests were used to compare two groups in each panel. NS, not significant (P ≥ 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
