Amplification of ColE1 related plasmids in recombinant cultures of Escherichia coli after IPTG induction.
ColE1-derived plasmids containing different recombinant genes which are controlled by the tac promoter were amplified following induction with IPTG, but no amplification occurred if product formation was not induced. The plasmid copy number of recombinant E. coli increased three- to sixfold within a period of about 6 h in shake flask experiments, batch cultures, and glucose-limited fed-batch cultivations. Plasmid amplification occurred in E. coli B strains as well as in K-12 strains with different plasmids (rop+ and rop-) coding for various heterologous proteins. The amplification was not caused by a toxic effect of IPTG, but was related to a strong inhibition of translation and chromosomal replication after the induction of heterologous gene expression. Similar to the amplification after chloramphenicol addition, plasmid replication proceeded even if oriC replication and translation were inhibited following strong induction of a recombinant gene. In accordance with the effect of chloramphenicol, the level of ppGpp, which is a negative regulator of ColE1 derived plasmid replication, decreased after induction.
Teich
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Martin-Luther-Universität Halle-Wittenberg, Fachbereich Biochemie/Biotechnologie, Institut für Biotechnologie, Halle, Germany.
Lin
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HY
Andersson
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Meyer
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S
Neubauer
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D001426:Bacterial Proteins / Q000235:genetics*; D003086:Bacteriocin Plasmids; D002701:Chloramphenicol / Q000494:pharmacology; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification / Q000187:drug effects*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects; D016601:RNA-Binding Proteins / Q000235:genetics*; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D003470:Culture Media; D016601:RNA-Binding Proteins; C054243:Rop protein, ColE1 plasmid; D007544:Isopropyl Thiogalactoside; D002701:Chloramphenicol