Fast and sensitive laser-based enzymatic detection of the lactose operon in microorganisms.
This work investigates the ability of laser-based, time-resolved fluorometry to detect the lactose operon genetic marker in microorganisms and to study protein-DNA interactions. In the first study, rapid detection of the Escherichia coli lacZY operon inserted in two strains of Pseudomonas proposed as fungal biological control organisms was achieved. Optimization of incubation time, immobilization apparatus size, and reagent volumes, along with the laser-based instrumentation, yielded an assay capable of detecting 10(4) immobilized lac+ Pseudomonas fluorescens cells within a 30-min incubation time. In the second study, the synthesis of E. coli beta-galactosidase was monitored in "real-time" with observable enzymatic activity beginning 4 to 5 min after induction with isopropylthiogalactoside.
Basile
F
F
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393.
Hughes
K D
KD
Wisniowski
P E
PE
Gorenstein
D G
DG
Lytle
F E
FE
McCay-Buis
T S
TS
Huber
D M
DM
Hemming
B C
BC
D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005470:Fluorometry; D005798:Genes, Bacterial; D005809:Genes, Regulator; D005818:Genetic Engineering; D005819:Genetic Markers; D007763:Lac Operon; D007834:Lasers; D011549:Pseudomonas / Q000235:genetics*; D012680:Sensitivity and Specificity; D013997:Time Factors; D001616:beta-Galactosidase / Q000096:biosynthesis
D004251:DNA Transposable Elements; D005819:Genetic Markers; D001616:beta-Galactosidase