PMID: 9547269Jun 20, 1998Paper

Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids

Nucleic Acids Research
S G WilliamsJulian A J Hanak

Abstract

The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages ...Continue Reading

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Citations

Nov 18, 2011·Applied Microbiology and Biotechnology·Filomena SilvaFernanda C Domingues
Apr 13, 2013·World Journal of Microbiology & Biotechnology·Xuejin ZhaoYuan Song
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