The application of multi-parameter flow cytometry to the study of recombinant Escherichia coli batch fermentation processes
- 13 July 2004
- journal article
- Published by Oxford University Press (OUP) in Journal of Industrial Microbiology & Biotechnology
- Vol. 31 (7), 311-322
- https://doi.org/10.1007/s10295-004-0151-8
Abstract
Multi-parameter flow cytometric techniques coupled with dual colour fluorescent staining were used to study the physical and metabolic consequences of inclusion body formation in batch cultures of the recombinant Escherichia coli strain MSD3735. This strain contains a plasmid coding for the isopropylthiogalactopyranoside-inducible model eukaryotic protein AP50. It is known that the synthesis of foreign proteins at high concentrations can exert a severe metabolic stress on the host cell and that morphological changes can occur. In this work, using various points of induction, it was shown that inclusion body formation is followed immediately by measurable changes in the characteristic intrinsic light scatter patterns for the individual cell (forward scatter, 90° side scatter) and a concomitant progressive change in the individual cell physiological state with respect to both cytoplasmic membrane polarisation and permeability. This work establishes flow cytometry as a potentially valuable tool for monitoring recombinant fermentation processes, providing important information for scale-up. Further, we discuss the possibility of optimising inclusion body formation by manipulating the fermentation conditions based on these rapid “real-time” measurements.Keywords
This publication has 23 references indexed in Scilit:
- Further studies related to the scale‐up of high cell density escherichia coli fed‐batch fermentations:Biotechnology & Bioengineering, 2003
- Study of the interaction of the medium chain μ2 subunit of the clathrin-associated adapter protein complex 2 with cytotoxic T-lymphocyte antigen 4 and CD28Biochemical Journal, 2001
- Substrate gradient formation in the large-scale bioreactor lowers cell yield and increases by-product formationBioprocess and Biosystems Engineering, 1998
- Hydrodynamics of Stirred BioreactorsApplied Mechanics Reviews, 1998
- Viability assessment of bacteria in mixed populations using flow cytometry*Journal of Microscopy, 1995
- Plasmid‐encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteriaBiotechnology & Bioengineering, 1990
- High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene productGene, 1989
- Single–Cell Light Scatter as a Probe of Refractile Body Formation in Recombinant Escherichia ColiNature Biotechnology, 1988
- Dissolved oxygen concentration profiles in a production‐scale bioreactorBiotechnology & Bioengineering, 1984
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970