Isotopomer distributions in amino acids from a highly expressed protein as a proxy for those from total protein

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{sup 13}C-based metabolic flux analysis provides valuable information about bacterial physiology. Though many biological processes rely on the synergistic functions of microbial communities, study of individual organisms in a mixed culture using existing flux analysis methods is difficult. Isotopomer-based flux analysis typically relies on hydrolyzed amino acids from a homogeneous biomass. Thus metabolic flux analysis of a given organism in a mixed culture requires its separation from the mixed culture. Swift and efficient cell separation is difficult and a major hurdle for isotopomer-based flux analysis of mixed cultures. Here we demonstrate the use of a single highly-expressed protein to analyze ... continued below

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Shaikh, Afshan; Shaikh, Afshan S.; Tang, Yinjie; Mukhopadhyay, Aindrila & Keasling, Jay D. June 27, 2008.

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{sup 13}C-based metabolic flux analysis provides valuable information about bacterial physiology. Though many biological processes rely on the synergistic functions of microbial communities, study of individual organisms in a mixed culture using existing flux analysis methods is difficult. Isotopomer-based flux analysis typically relies on hydrolyzed amino acids from a homogeneous biomass. Thus metabolic flux analysis of a given organism in a mixed culture requires its separation from the mixed culture. Swift and efficient cell separation is difficult and a major hurdle for isotopomer-based flux analysis of mixed cultures. Here we demonstrate the use of a single highly-expressed protein to analyze the isotopomer distribution of amino acids from one organism. Using the model organism E. coli expressing a plasmid-borne, his-tagged Green Fluorescent Protein (GFP), we show that induction of GFP does not affect E. coli growth kinetics or the isotopomer distribution in nine key metabolites. Further, the isotopomer labeling patterns of amino acids derived from purified GFP and total cell protein are indistinguishable, indicating that amino acids from a purified protein can be used to infer metabolic fluxes of targeted organisms in a mixed culture. This study provides the foundation to extend isotopomer-based flux analysis to study metabolism of individual strains in microbial communities.

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  • Journal Name: Analytical Chemistry; Journal Volume: 80; Related Information: Journal Publication Date: 2008

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  • Report No.: LBNL-693E
  • Grant Number: DE-AC02-05CH11231
  • DOI: 10.1021/ac071445+ | External Link
  • Office of Scientific & Technical Information Report Number: 934964
  • Archival Resource Key: ark:/67531/metadc901000

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  • June 27, 2008

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  • Sept. 27, 2016, 1:39 a.m.

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  • Sept. 30, 2016, 6:38 p.m.

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Shaikh, Afshan; Shaikh, Afshan S.; Tang, Yinjie; Mukhopadhyay, Aindrila & Keasling, Jay D. Isotopomer distributions in amino acids from a highly expressed protein as a proxy for those from total protein, article, June 27, 2008; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc901000/: accessed November 14, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.