Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter |
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Authors: | J E Larsen K Gerdes J Light S Molin |
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Institution: | Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark Tel. 45-9-158600, ext.2428 |
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Abstract: | By insertion of a DNA fragment, containing the phage lambda pR promoter and the pM-promoted cI857 allele of the lambda repressor gene, in plasmid R1 upstream of the replication control genes, cloning vectors have been constructed which are present in one copy per chromosome at temperatures below 37 degrees C, and which display uncontrolled replication at 42 degrees C. Derivatives have been made which carry the R1 par region, stabilizing the plasmid at low temperature when grown in the absence of selection pressure. Cells harbouring these plasmids stop growing after 1-2 h incubation at 42 degrees C, and at this time 50% of the total DNA in the cells is plasmid DNA corresponding to more than 1000 plasmid molecules per cell. Concomitant with plasmid amplification at the high temperature, synthesis of plasmid-coded gene products is amplified, and these vectors can therefore be utilized for obtaining greatly enhanced yields of gene products that may be detrimental to the host cell when present in large amounts. |
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Keywords: | Conditional runaway-replication gene amplification cloning vehicle detrimental gene products recombinant DNA bp base pairs EtBr ethidium bromide kb kilobase pairs LA LB media see MATERIALS section b METHODS section b [ ] indicates plasmid-carrier state |
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