Fig. 3

Protein secretion through the truncated FT3SS can be both controlled and improved. E. coli MC1000 ΔfliC ΔflgKL (ΔCKL or ‘prototype’), ΔflhDC, ΔfliC ΔflgKL ΔflgDE (ΔCKL-DE) or ΔfliC ΔflgKL ΔclpX (ΔCKL-X or ‘Mark II strain’) containing pTrc-FliC-ΔD3 was grown with 0.05 mM IPTG (or 1 mM to allow overexpression in ΔflhDC) and harvested at OD₆₀₀ 1.0. Secreted and intracellular cell fractions were loaded for SDS-PAGE in the quantities described in Fig. 2. A FliC-ΔD3 protein standard was included to allow quantification. Samples underwent a Coomassie staining or immunoblot analysis of cells and supernatant using b anti-flagellin (αH48) or c anti(α)-GroEL primary antibodies and a HRP-conjugated secondary