Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration.
We report the construction of two novel Escherichia coli strains (DH1lacdapD and DH1lacP2dapD) that facilitate the antibiotic-free selection and stable maintenance of recombinant plasmids in complex media. They contain the essential chromosomal gene, dapD, under the control of the lac operator/promoter. Unless supplemented with IPTG (which induces expression of dapD) or DAP, these cells lyse. However, when the strains are transformed with a multicopy plasmid containing the lac operator, the operator competitively titrates the LacI repressor and allows expression of dapD from the lac promoter. Thus transformants can be isolated and propagated simply by their ability to grow on any medium by repressor titration selection. No antibiotic resistance genes or other protein expressing sequences are required on the plasmid, and antibiotics are not necessary for plasmid selection, making these strains a valuable tool for therapeutic DNA and recombinant protein production. We describe the construction of these strains and demonstrate plasmid selection and maintenance by repressor titration, using the new pORT plasmid vectors designed to facilitate recombinant DNA exploitation.
Cranenburgh
R M
RM
Cobra Therapeutics Ltd, The Science Park, Keele, Staffordshire ST5 5SP, UK.
Hanak
J A
JA
Williams
S G
SG
Sherratt
D J
DJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005819:Genetic Markers; D007612:Kanamycin / Q000494:pharmacology; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D014170:Transformation, Genetic
D005819:Genetic Markers; D007612:Kanamycin