Dual-origin plasmid vectors whose origin of replication is controlled by the coliphage lambda promoter pL

• Published in: Gene Vol. 28; no. 3; pp. 293 - 300
• Main Authors: Yarranton, G.T., Wright, E., Robinson, M.K., Humphreys, G.O.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 01.01.1984; Amsterdam Elsevier; New York, NY
• Subjects: Bacteriocin Plasmids; Bacteriophage lambda - genetics; Biological and medical sciences; Biotechnology; ColE1; DNA Replication; DNA, Bacterial - genetics; DNA, Recombinant - analysis; DNA, Viral - genetics; E. coli; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Genetic engineering; Genetic technics; Genetic Vectors; Methods. Procedures. Technologies; Operon; Plasmids; Recombinant DNA; RNA - genetics; RNA II primer; RNA, Bacterial - genetics; temperature-controlled copy number; Vectors (cloning, transfer, expression). Insertion sequences and transposons; λ repressor; λ repressor; Km; LB, Mg; temperature-controlled copy number; ColE1; Cm; Recombinant DNA; the superscript S; RNA II; bp; the superscript R; Ap; TCA; Δ; RNA II primer; kb; p L; E. coli; EtBr; Origin; Temperature; Bactériophage lambda; Induction; Escherichia coli; Transcription repressor; Gene expression; Virus; Lambda phage group; Gene amplification; Regulation(control); Transcription promotor; Styloviridae; Bacteria; Bactériophage; Replication; Escherichieae; Vector; Enterobacteriaceae
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(84)90146-X

The insertion of an XhoI linker 30 bp from the 5' end of the RNA II primer for ColE1 plasmid replication has allowed us to replace the natural RNA II promoter by other controllable promoters, in particular λ p l . Manipulation of this modified origin was facilitated by constructing dual-origin plasmids, stable maintenance being directed by the pSC101 origin. Experiments showed that such dual-origin plasmids could be stably maintained at approximately four copies per chromosome at 30° C and readily amplified by thermal induction of strains carrying a thermolabile λ repressor. The use of such plasmids for the construction of stable, amplifiable expression vectors is discussed.