ISSN:
0006-3592
Keywords:
flow cytometry
;
recombinant E. coli
;
β-galactosidase assay
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
A flow cytometric method was developed for the assay of β-galactosidase in single Escherichia coli cells. A new fluorogenic substrate for β-galactosidase, C12FDG, contains a lipophilic group that allows the substrate to penetrate through cell membranes under normal conditions. When the substrate is hydrolyzed by intracellular β-galactosidase, a green fluorescent product is formed and retained inside the cell. Consequently, the stained β-galactosidase-positive cells exhibit fluorescence, which is detected by flow cytometry. This new assay was used to analyze the segregational instability caused by a reduction in specific growth rate of the plasmid-bearing cells in the T7 expression system. Induction results in a substantial accumulation of intracellular β-galactosidase along with a rapid increase in the fraction of plasmid-free cells. Once the cells lose the plasmid, they no longer produce β-galactosidase, which is reduced by at least half every generation; thus, after staining, the fluorescent, plasmid-bearing cells can be distinguished from the nonfluorescent, plasmid-free cells using flow cytometry. This article describes the feasibility of the flow cytometric assay for single E. coli cells and reports the optimal assay conditions. A direct relationship between β-galactosidase activity and green fluorescence intensity was found, and the fractions of recombinant cells in batch cultures were analyzed after various levels of induction. © 1993 John Wiley & Sons, Inc.
Additional Material:
9 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/bit.260420605
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