New bioreactor-coupled rapid stopped-flow sampling technique for measurements of metabolite dynamics on a subsecond time scale.
Knowledge of concentrations of intracellular metabolites is important for quantitative analysis of metabolic networks. As far as the very fast response of intracellular metabolites in the millisecond range is concerned, the frequently used pulse technique shows an inherent limitation. The time span between the disturbance and the first sample is constrained by the time necessary to obtain a homogeneous distribution of the pertubation within the bioreactor. For determination of rapid changes, a novel sampling technique based on the stopped-flow method has been developed. A continuous stream of biosuspension leaving the bioreactor is being mixed with a glucose solution in a turbulent mixing chamber. Through computer-aided activation of sequentially positioned three-way valves, different residence times and thus reaction times can be verified. The application of this new sampling method is illustrated with examples including measurements of adenine nucleotides and glucose-6-phosphate in Saccharomyces cerevisiae as well as measurements related to the PTS system in Escherichia coli.
Buziol
Stefan
S
Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Bashir
Imtiaz
I
Baumeister
Anja
A
Claassen
Wolfgang
W
Noisommit-Rizzi
Naruemol
N
Mailinger
Werner
W
Reuss
Matthias
M
D000227:Adenine Nucleotides / Q000032:analysis / Q000378:metabolism; D019149:Bioreactors; D002478:Cells, Cultured; D003470:Culture Media / Q000378:metabolism*; D004867:Equipment Design; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D005947:Glucose / Q000378:metabolism*; D007218:Industrial Microbiology / Q000295:instrumentation* / Q000379:methods; D007700:Kinetics; D010728:Phosphoenolpyruvate; D019289:Pyruvic Acid / Q000032:analysis / Q000378:metabolism; D015203:Reproducibility of Results; D012212:Rheology / Q000295:instrumentation* / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors
D000227:Adenine Nucleotides; D003470:Culture Media; D010728:Phosphoenolpyruvate; D019289:Pyruvic Acid; D005947:Glucose