Effects of plasmid copy number and runaway plasmid replication on overproduction and excretion of beta-lactamase from Escherichia coli.
Runaway plasmid replication can be used to increase target gene dosage and thereby overproduce proteins within the bacterium Escherichia coli. However, the presence of excessive plasmid DNA often alters normal cell functions. High copy number plasmids with strong promoters place a severe metabolic burden on the cell, causing a decreased specific growth rate and changes in cell physiology. Induction of beta-lactamase synthesis from the tac promoter on plasmid pKN causes runaway plasmid replication and excretion of beta-lactamase. Runaway plasmid replication results from readthrough of tac promoter transcripts into the replication region of the plasmid. Both high plasmid copy numbers and a strong promoter (tac) are necessary to achieve the level of overproduction necessary for excretion of beta-lactamase, but high-level target protein synthesis is detrimental to the cell. A derivative of pKN which is more easily regulated was constructed by adding the lacI gene to the plasmid.
Togna
A P
AP
School of Chemical Engineering, Cornell University, Ithaca, New York 14853.
Shuler
M L
ML
Wilson
D B
DB
D004261:DNA Replication; D004587:Electrophoresis, Agar Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007544:Isopropyl Thiogalactoside; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics
D007544:Isopropyl Thiogalactoside; D001618:beta-Lactamases