Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids.
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.
Williams
S G
SG
Cobra Therapeutics Limited, The Science Park, University of Keele, Keele, Staffordshire ST5 5SP, UK.
Cranenburgh
R M
RM
Weiss
A M
AM
Wrighton
C J
CJ
Sherratt
D J
DJ
Hanak
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D004794:Enzyme Repression; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D007613:Kanamycin Resistance / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism*; D012641:Selection, Genetic; D017186:Titrimetry; D014169:Transformation, Bacterial
D012097:Repressor Proteins