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