Rapid sampling for analysis of in vivo kinetics using the BioScope: a system for continuous-pulse experiments.
In this article we present a novel device, the BioScope, which allows elucidation of in vivo kinetics of microbial metabolism via perturbation experiments. The perturbations are carried out according to the continuous-flow method. The BioScope consists of oxygen permeable silicon tubing, connected to the fermentor, through which the broth flows at constant velocity. The tubing has a special geometry (serpentine channel) to ensure plug flow. After leaving the fermentor, the broth is mixed with a small flow of perturbing agent. This represents the start of the perturbation. The broth is sampled at different locations along the tubing, corresponding to different incubation times. The maximal incubation time is 69 s; the minimally possible time interval between the samples is 3-4 s. Compared to conventional approaches, in which the perturbation is carried out in the fermentor, the BioScope offers a number of advantages. (1) A large number of different perturbation experiments can be carried out on the same day, because the physiological state of the fermentor is not perturbed. (2) In vivo kinetics during fed-batch experiments and in large-scale reactors can be investigated. (3) All metabolites of interest can be measured using samples obtained in a single experiment, because the volume of the samples is unlimited. (4) The amount of perturbing agent spent is minimal, because only a small volume of broth is perturbed. (5) The system is completely automated. Several system properties, including plug-flow characteristics, mixing, oxygen and carbon dioxide transfer rates, the quenching time, and the reproducibility have been explored, with satisfactory results. Responses of several glycolytic intermediates in Saccharomyces cerevisiae to a glucose pulse, measured using a conventional approach are compared to results obtained with the BioScope. The agreement between the results demonstrates that the BioScope is indeed a promising device for studying in vivo kinetics.
Visser
Diana
D
Puroc, P.O. Box 21, 4200AA Gorinchem, The Netherlands. d.visser@puroc.com
van Zuylen
Gertan A
GA
van Dam
Jan C
JC
Oudshoorn
Arthur
A
Eman
Michael R
MR
Ras
Cor
C
van Gulik
Walter M
WM
Frank
Johannes
J
van Dedem
Gijs W K
GW
Heijnen
Joseph J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D019149:Bioreactors; D004867:Equipment Design; D005285:Fermentation; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D005947:Glucose / Q000378:metabolism*; D006019:Glycolysis; D008567:Membranes, Artificial; D010100:Oxygen / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D018401:Sample Size; D012680:Sensitivity and Specificity
D008567:Membranes, Artificial; D000255:Adenosine Triphosphate; D005947:Glucose; D010100:Oxygen