title: FIBS-enabled noninvasive metabolic profiling.
abstract: In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on Förster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.
authors: Centre for Process Systems Engineering, Department of Chemical Engineering and Chemical Technology, Imperial College London.
authors: Alireza
authors: A
authors: Behjousiar
authors: Antony
authors: A
authors: Constantinou
authors: Karen M
authors: KM
authors: Polizzi
authors: Cleo
authors: C
authors: Kontoravdi
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods* / Q000592:standards; D016466:CHO Cells; D002138:Calibration; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods* / Q000592:standards; D055442:Metabolome; D055432:Metabolomics / Q000379:methods* / Q000592:standards