Fig. 5.

Screening for strains which are high capacity secretors of recombinant cutinase, and additional substrates. a The ‘prototype’ E. coli MC1000 ΔfliC ΔflgKL (ΔCKL) strain with additional combinations of: ΔmotAB (mot) ΔflgMN (MN) ΔfliDST (DST) or ΔclpX (X), which contained pJex-fliC47-cutinase were grown with 0.05 mM IPTG and harvested at OD₆₀₀ 1.0. Secreted fractions (Sn) were analysed by the MUB florescence assay as described in Fig. 4c. Results from three biological replicates were normalised to ΔCKL, ± SE shown, one-way ANOVA: p ≤ 0.001. Tukey’s multiple comparison test to ΔCKL: *p ≤ 0.05, **p ≤ 0.01. Representative immunoblot of the intracellular fraction from 15 µL cell culture shown below. b E. coli ΔCKL (‘prototype’), ΔCKL-X-mot (Mark III strain) and ΔCKL-mot-DST expressing pJex-fliC47-CH₂ (+, upper), pTrc-FliC-ΔD3 (+, lower), or empty vector (−) were grown (as above) and prepared for immunoblot using either an anti-FLAG-HRP (αFLAG) antibody (upper) or an anti-flagellin (αH48) antibody and a HRP conjugated secondary (lower). The equivalent of 15 µL cell culture was loaded for intracellular fractions, and either 300 or 60 µL for the secreted fractions (for CH₂ and FliC-ΔD3 respectively), along with the relevant protein standard to allow quantification. c Whole cell fractions underwent immunoblot with an anti-FlhA antibody (αFlhA), results from densitometry are presented relative to ΔCKL. Five biological repeats, ± SE and individual data points shown. One-way ANOVA: p < 0.005 and Tukey’s multiple comparison test: *p < 0.05, **p < 0.01