Quasi-continuous combined scattered light and fluorescence measurements: a novel measurement technique for shaken microtiter plates.
A novel quasi-continuous on-line measuring technique for shaken microtiter plates is presented. Light scattering as well as intracellular and/or protein fluorescence (e.g. NADH, YFP) is measured during the shaking procedure, thus allowing a process monitoring of 96 different simultaneous cultures in a microtiter plate. In contrast to existing measurement techniques, the shaking process does not have to be stopped to take the measurements, thus avoiding the corresponding interruption of the cultures' oxygen supply and any unpredictable effects on the cultures. Experiments were conducted with E. coli in LB, TB, and MOPS minimal medium and V. natriegens in modified LB and TB media. Intensity curves of scattered light and NADH fluorescence were used to distinguish different lag phases, growth velocities, or inoculation densities. Data from this new method corresponded well to the off-line measured optical densities and to the oxygen transfer rates of cultures run in simultaneously conducted shake flask experiments at equivalent oxygen transfer capacities. With the aid of yellow fluorescence protein fused to interleukin-6 the optimal induction time of an expressing E. coli strain could be determined by on-line monitoring of product formation. Thus, this measuring technique enables the researcher to evaluate and to discriminate different cultures on a screening level and to improve screening conditions, process development and scale-up.
Department of Biochemical Engineering, RWTH Aachen University, Sammelbau Biologie, Worringer Weg 1, D-52074 Aachen, Germany.
M
M
Samorski
G
G
Müller-Newen
J
J
Büchs
D001426:Bacterial Proteins / Q000378:metabolism; D015374:Biosensing Techniques; D001709:Biotechnology / Q000295:instrumentation* / Q000379:methods; D002138:Calibration; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D004926:Escherichia coli / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D009243:NAD / Q000378:metabolism; D055095:Optics and Photonics; D010100:Oxygen / Q000378:metabolism; D012542:Scattering, Radiation; D013997:Time Factors; D014733:Vibrio / Q000378:metabolism
D001426:Bacterial Proteins; D015850:Interleukin-6; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D009243:NAD; D010100:Oxygen