Automated Sampling Device for Monitoring Intracellular Metabolite Dynamics

• Published in: Analytical biochemistry Vol. 270; no. 1; pp. 88 - 96
• Main Authors: Schaefer, U., Boos, W., Takors, R., Weuster-Botz, D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-05-1999
• Subjects: automated sampling device; Biosensing Techniques; Centrifugation; Cold Temperature; Escherichia coli; Escherichia coli - metabolism; Fructosediphosphates - metabolism; Glucose - metabolism; glucose shift; high sampling rate; Kinetics; metabolite oscillations; methanol spraying; Models, Chemical; Specimen Handling - instrumentation; stirred tank reactor; automated sampling device; methanol spraying; stirred tank reactor; metabolite oscillations; Escherichia coli; high sampling rate; glucose shift
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1999.4048

An automated sampling device coupled to a stirred tank reactor was developed for monitoring intracellular metabolite dynamics. Sample flasks fixed in transport magazines were moved by a step engine in a way that each sample flask was filled within 220 ms, resulting in a sampling rate of 4.5 s−1. Rapid inactivation of the metabolism was achieved by spraying the samples into 60% methanol at −50°C. After centrifugation of the quenched cells at −20°C the metabolites were extracted with perchloric acid and analyzed biochemically or with HPLC. The automated sampling device was applied for investigation of the intracellular metabolite dynamics of glycolysis inEscherichia coliafter rapid glucose addition to a glucose-limited steady-state culture. For the first time oscillations of intracellular metabolite concentrations like glucose-6-phosphate, phosphoenolpyruvate, glyceraldehyde 3-phosphate, dihydroxyacetonphosphate, 3-phosphoglycerate, and pyruvate were quantified on a subseconds to seconds scale inE. coli.As an example, the kinetics of the decomposition of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetonphosphate were investigated by use of a well-known mechanistic kinetic model and the measuredin vivometabolite dynamics.