Print Email Facebook Twitter Microscale quantitative analysis of polyhydroxybutyrate in prokaryotes using IDMS Title Microscale quantitative analysis of polyhydroxybutyrate in prokaryotes using IDMS Author Velasco Alvarez, M.I. (TU Delft OLD BT/Cell Systems Engineering) ten Pierick, A. (Vrije Universiteit Amsterdam) van Dam, P.T.N. (TU Delft OLD BT/Cell Systems Engineering) Maleki Seifar, R. (TU Delft OLD BT/Cell Systems Engineering) van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology) Wahl, S.A. (TU Delft OLD BT/Cell Systems Engineering) Date 2017 Abstract Poly(3-hydroxybutyrate) (PHB) is an interesting biopolymer for replacing petroleum-based plastics, its biological production is performed in natural and engineered microorganisms. Current metabolic engineering approaches rely on high-throughput strain construction and screening. Analytical procedures have to be compatible with the small scale and speed of these approaches. Here, we present a method based on isotope dilution mass spectrometry (IDMS) and propanolysis extraction of poly(3-hydroxybutyrate) from an Escherichia coli strain engineered for PHB production. As internal standard (IS), we applied an uniformly labeled 13C-cell suspension, of an E. coli PHB producing strain, grown on U-13C-glucose as C-source. This internal 13C-PHB standard enables to quantify low concentrations of PHB (LOD of 0.01 µg/gCDW) from several micrograms of biomass. With this method, a technical reproducibility of about 1.8% relative standard deviation is achieved. Furthermore, the internal standard is robust towards different sample backgrounds and dilutions. The early addition of the internal standard also enables higher reproducibility and increases sensitivity and throughput by simplified sample preparation steps Subject polyhydroxybutyrate3-hydroxy butyrateGC-MS13C-labeled internal standardisotope dilutionEscherichia coliOA-Fund TU Delft To reference this document use: http://resolver.tudelft.nl/uuid:1430de82-5f33-42f3-ad10-066f2f2b2086 DOI https://doi.org/10.3390/metabo7020019 ISSN 2218-1989 Source Metabolites, 7 (2) Part of collection Institutional Repository Document type journal article Rights © 2017 M.I. Velasco Alvarez, A. ten Pierick, P.T.N. van Dam, R. Maleki Seifar, Mark C.M. van Loosdrecht, S.A. Wahl Files PDF metabolites_07_00019.pdf 1.13 MB Close viewer /islandora/object/uuid:1430de82-5f33-42f3-ad10-066f2f2b2086/datastream/OBJ/view