Catching prompt metabolite dynamics in Escherichia coli with the BioScope at oxygen rich conditions

The design and application of a BioScope, a mini plug-flow reactor for carrying out pulse response experiments, specifically designed for Escherichia coli is presented. Main differences with the previous design are an increased volume-specific membrane surface for oxygen transfer and significantly decreased sampling intervals. The characteristics of the new device (pressure drop, residence time distribution, plug-flow behavior and O2 mass transfer) were determined and evaluated. Subsequently, 2.8mM glucose perturbation experiments on glucose-limited aerobic E. coli chemostat cultures were carried out directly in the chemostat as well as in the BioScope (for two time frames: 8 and 40s). It was ensured that fully aerobic conditions were maintained during the perturbation experiments. To avoid metabolite leakage during quenching, metabolite quantification (glycolytic and TCA-cycle intermediates and nucleotides) was carried out with a differential method, whereby the amounts measured in the filtrate were subtracted from the amounts measured in total broth. The dynamic metabolite profiles obtained from the BioScope perturbations were very comparable with the profiles obtained from the chemostat perturbation. This agreement demonstrates that the BioScope is a promising device for studying in vivo kinetics in E. coli that shows much faster response (<10s) in comparison with eukaryotes.