Bacterial expression system with tightly regulated gene expression and plasmid copy number.
A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number. Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter. gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.
Bowers
Lisa M
LM
Department of Bacteriology, University of Wisconsin-Madison, 420 Henry Mall Room 151, 1550 Linden Drive, Madison, WI 53706-1567, USA.
Lapoint
Kathleen
K
Anthony
Larry
L
Pluciennik
Anna
A
Filutowicz
Marcin
M
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001089:Arabinose / Q000494:pharmacology; D015153:Blotting, Western; D003087:Colicins / Q000235:genetics; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D018741:Replication Origin / Q000235:genetics; D017422:Sequence Analysis, DNA; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D003087:Colicins; D029968:Escherichia coli Proteins; C506744:rep protein, E coli; D001089:Arabinose; D001616:beta-Galactosidase; D000251:Adenosine Triphosphatases; D004265:DNA Helicases