Nucleic Acids Research, Vol 26, Issue 9 2120-2124, Copyright © 1998 by Oxford University Press
SG Williams, RM Cranenburgh, AM Weiss, CJ Wrighton, DJ Sherratt and JA J Hanak
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 for use as gene therapy vectors. Regulation in the same
way of an essential, endogenous bacterial gene will allow the production of
recombinant therapeutics devoid of residual antibiotic contamination.
ARTICLES
Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids
Cobra Therapeutics Limited, The Science Park, University of Keele, Keele, Staffordshire ST5 5SP, UK.
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