Flow cytometry as a useful tool for process development: Rapid evaluation of expression systems

Anant Patkar; Natarajan Vijayasankaran; Dan W. Urry; Friedrich Srienc

doi:10.1016/S0168-1656(01)00399-6

Flow cytometry as a useful tool for process development: Rapid evaluation of expression systems

Anant Patkar, Natarajan Vijayasankaran, Dan W. Urry, Friedrich Srienc

Biotechnology Institute

Research output: Contribution to journal › Article › peer-review

31 Scopus citations

Abstract

Flow cytometry is an established tool in fundamental studies of single-cell microbial physiology. Here we show that it can also provide valuable information for process development. Using recombinant Escherichia coli strains, which express the protein-based polymer (GVGIP)₂₆₀GVGVP, the utility of flow cytometry in monitoring and optimization of fermentations is demonstrated. Single cell right angle light scatter was found to be significantly affected by intracellular product formation possibly due to the formation of inclusion bodies. Translational fusions with green fluorescent protein (GFP) enabled monitoring of product accumulation, as well as plasmid free cell fraction (PFCF). Such fusions also allowed rapid evaluation of induction strategies and three different expression systems based on the T7 promoter, T7-lac promoter and the P_BAD promoter. The expression system based on the P_BAD promoter was found to be superior to the T7-based system.

Original language	English (US)
Pages (from-to)	217-229
Number of pages	13
Journal	Journal of Biotechnology
Volume	93
Issue number	3
DOIs	https://doi.org/10.1016/S0168-1656(01)00399-6
State	Published - Feb 28 2002

Bibliographical note

Funding Information:
We thank the Office of Naval Research for supporting this work. Dr Xu and Dr McPherson of Bioelastics Research Limited (BRL), Birmingham, AL, provided valuable assistance in this study.

Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.

Keywords

Escherichia coli
Flow cytometry
GFP

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title = "Flow cytometry as a useful tool for process development: Rapid evaluation of expression systems",

abstract = "Flow cytometry is an established tool in fundamental studies of single-cell microbial physiology. Here we show that it can also provide valuable information for process development. Using recombinant Escherichia coli strains, which express the protein-based polymer (GVGIP)260GVGVP, the utility of flow cytometry in monitoring and optimization of fermentations is demonstrated. Single cell right angle light scatter was found to be significantly affected by intracellular product formation possibly due to the formation of inclusion bodies. Translational fusions with green fluorescent protein (GFP) enabled monitoring of product accumulation, as well as plasmid free cell fraction (PFCF). Such fusions also allowed rapid evaluation of induction strategies and three different expression systems based on the T7 promoter, T7-lac promoter and the PBAD promoter. The expression system based on the PBAD promoter was found to be superior to the T7-based system.",

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author = "Anant Patkar and Natarajan Vijayasankaran and Urry, {Dan W.} and Friedrich Srienc",

note = "Funding Information: We thank the Office of Naval Research for supporting this work. Dr Xu and Dr McPherson of Bioelastics Research Limited (BRL), Birmingham, AL, provided valuable assistance in this study. Copyright: Copyright 2008 Elsevier B.V., All rights reserved.",

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AU - Vijayasankaran, Natarajan

AU - Urry, Dan W.

AU - Srienc, Friedrich

N1 - Funding Information: We thank the Office of Naval Research for supporting this work. Dr Xu and Dr McPherson of Bioelastics Research Limited (BRL), Birmingham, AL, provided valuable assistance in this study. Copyright: Copyright 2008 Elsevier B.V., All rights reserved.

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N2 - Flow cytometry is an established tool in fundamental studies of single-cell microbial physiology. Here we show that it can also provide valuable information for process development. Using recombinant Escherichia coli strains, which express the protein-based polymer (GVGIP)260GVGVP, the utility of flow cytometry in monitoring and optimization of fermentations is demonstrated. Single cell right angle light scatter was found to be significantly affected by intracellular product formation possibly due to the formation of inclusion bodies. Translational fusions with green fluorescent protein (GFP) enabled monitoring of product accumulation, as well as plasmid free cell fraction (PFCF). Such fusions also allowed rapid evaluation of induction strategies and three different expression systems based on the T7 promoter, T7-lac promoter and the PBAD promoter. The expression system based on the PBAD promoter was found to be superior to the T7-based system.

AB - Flow cytometry is an established tool in fundamental studies of single-cell microbial physiology. Here we show that it can also provide valuable information for process development. Using recombinant Escherichia coli strains, which express the protein-based polymer (GVGIP)260GVGVP, the utility of flow cytometry in monitoring and optimization of fermentations is demonstrated. Single cell right angle light scatter was found to be significantly affected by intracellular product formation possibly due to the formation of inclusion bodies. Translational fusions with green fluorescent protein (GFP) enabled monitoring of product accumulation, as well as plasmid free cell fraction (PFCF). Such fusions also allowed rapid evaluation of induction strategies and three different expression systems based on the T7 promoter, T7-lac promoter and the PBAD promoter. The expression system based on the PBAD promoter was found to be superior to the T7-based system.

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Flow cytometry as a useful tool for process development: Rapid evaluation of expression systems

Abstract

Bibliographical note

Keywords

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