Fig. 2.

Optimisation of cell culture induction and harvesting procedure for the FliC-ΔD3 protein secretion assay. The ‘prototype’ E. coli MC1000 ΔfliC ΔflgKL (ΔCKL) containing the plasmid pTrc-FliC-ΔD3 was grown in 100 mL flask cultures in LB and either a supplemented with 0.05 mM IPTG and harvested every hour or b supplemented with 0, 0.05, 0.1, 0.5 or 1 mM IPTG and harvested at OD₆₀₀ 1.0. FliC-ΔD3 was detected by immunoblot as described in Fig. 1a. Samples which represented either 25 µL or 300 µL culture media were loaded for SDS-PAGE for intracellular and secreted protein respectively. Densitometry analysis allowed quantification of secreted protein, throughout the growth curve. This was repeated to ensure that this corroborated with the general trend