Screening for highly active plasmid promoters via fusion to beta-galactosidase gene.
A plasmid containing promoter-deleted inactive beta-galactosidase gene [1] was used to select promoters of the pEP 121 plasmid [2]. Colonies of cells harboring reactivated beta-galactosidase gene were identified by their red color on McConkey plates. The quantitative amounts of beta-galactosidase produced in each clone were estimated by assaying enzyme activity and by measuring the specific beta-galactosidase protein following fractionation of total cells' proteins on polyacrylamide gel. A wide range of enzyme activities was observed. The most active promoter isolated was shown to promote beta-galactosidase production more efficiently, compared with the original beta-galactosidase promoter, amounting to 20% of all cell proteins. Such highly active promoters may be utilized in the future, to promote expression of cloned genes in bacteria.
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005696:Galactosidases / Q000235:genetics*; D005796:Genes; D009693:Nucleic Acid Hybridization; D009876:Operon; D010957:Plasmids; D001616:beta-Galactosidase / Q000235:genetics* / Q000302:isolation & purification
D005696:Galactosidases; D001616:beta-Galactosidase