Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids
| Autor: | Christopher J. Wrighton, Amanda Weiss, Julian A. J. Hanak, Rocky M. Cranenburgh, David J. Sherratt, Steven Geraint Williams |
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| Rok vydání: | 1998 |
| Předmět: |
Operator (biology)
Kanamycin Resistance Molecular Sequence Data Repressor lac operon Biology Lac repressor Marker gene Plasmid maintenance Plasmid Genetics Escherichia coli Amino Acid Sequence Cloning Molecular Selection Genetic Selectable marker Base Sequence Models Genetic Titrimetry Gene Expression Regulation Bacterial Chromosomes Bacterial Molecular biology Repressor Proteins Lac Operon bacteria Transformation Bacterial Enzyme Repression Plasmids Research Article |
| Zdroj: | Nucleic acids research. 26(9) |
| ISSN: | 0305-1048 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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