The application of multi-parameter flow cytometry to the study of recombinant Escherichia coli batch fermentation processes.
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 degrees 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.
Lewis
Gareth
G
School of Engineering (Chemical Engineering), The University of Birmingham, B15 2TT, Edgbaston, UK.
Taylor
Ian W
IW
Nienow
Alvin W
AW
Hewitt
Christopher J
CJ
D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D033983:Adaptor Protein Complex mu Subunits / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005434:Flow Cytometry; D005453:Fluorescence; D015964:Gene Expression Regulation, Bacterial; D002479:Inclusion Bodies / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods
D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C438885:adaptor protein complex 2, mu 1 subunit