Fig. 8

Cutinase expression and secretion: comparing the prototype, and most improved, strains and secretion constructs. The E. coli ‘prototype’ ΔfliC ΔflgKL (ΔCKL) or ‘Mark III strain’ ΔfliC ΔflgKL ΔclpX ΔmotAB (ΔCKL-X-mot) expressing either the prototype (SC0), improved (SC1) modular secretion vector, or pJex–fliC47-empty (empty), were grown as described in Fig. 3. Note that ΔCKL-X-mot expressing SC1 is our ‘Mark IV strain’. Both intracellular and secreted fractions underwent immunoblot analysis using either, an anti-FLAG-HRP (αFLAG) antibody to detect a intracellular and b secreted cutinase, or c anti-GroEL and a HRP secondary antibody to detect cytoplasmic protein contamination. Samples representing 15 μL and 300 μL cell culture were loaded for intracellular and supernatant samples, respectively. d Densitometry analysis was carried out on αFLAG probed, secreted fractions (Fig. 8b: representative image) of three biological replicates, normalised to ΔCKL-SC0, ± SE and individual data points shown. One-way ANOVA, p < 0.005 and Tukey’s multiple comparison test (to ΔCKL-SC0): ***p < 0.005, **p < 0.01, *p < 0.05. e Supernatant was also prepared for MUB protein secretion assay as described in Fig. 4c. Six biological replicates, normalised to ΔCKL-SC0, ± SE and individual data points shown. Two-way ANOVA, p < 0.001 and Tukey’s multiple comparison test (to ΔCKL-SC0): ****p < 0.001