Fig. 4

Development of a high throughput fluorescence assay to measure protein secretion through the truncated FT3SS. The ‘prototype’ E. coli MC1000 ΔfliC ΔflgKL (ΔCKL) containing either pJex-fliC47-cutinase or pJex-fliC47-empty were grown as described in Fig. 3 and prepared for a immunoblot with the anti-FLAG-HRP (αFLAG) antibody (where samples representing either 15 µL or 300 µL culture media for intracellular and secreted protein respectively were loaded onto the SDS-PAGE) (b) or florescence assay: 40 µL supernatant was added to 160 µL MUB substrate in a 96 well plate. Following incubation for 30 min at 30 °C, samples were visualised under UV light. c, d E. coli strains ΔfliC ΔflgKL (ΔCKL or ‘prototype’), ΔfliC ΔflgKL ∆clpX (ΔCKL-X or ‘Mark II strain’), ∆flhDC and ΔfliC ΔflgKL ∆flgDE (ΔCKL-DE) harbouring the cutinase expressing or empty vector plasmid were grown and c prepared for MUB secretion assay as described above; however following incubation, fluorescence was measured in a plate reader (excitation 302 nm, emission 446 nm). Results from one biological replicate, with three technical repeats. ± SE and individual data points shown. Two-way ANOVA (variables: strain and plasmid) and Tukey’s multiple comparison test: ****p < 0.001. d Representative immunoblot of secreted and intracellular fractions prepared from the same cell cultures