Stability of a host-vector system based on complementation of an essential gene in Escherichia coli.
Antibiotic selection is the most common selection system for plasmid-containing bacteria. This technique, nevertheless, can be a source of problems during the expression of heterologous genes in Escherichia coli. We have developed an alternative selection system based on the complementation of a chromosomal auxotrophic (dapD2) mutation by the corresponding wild type gene carried on a plasmid. We show that the system effectively selects for the presence of plasmid on solid and liquid medium. In addition, we have observed a loss of viability associated with high levels of gene expression and accumulation of a heterologous protein, but the selective power and improved intrinsic stability of the dap+ plasmid, compared to a beta-lactamase (bla) based vector, excludes overgrowth of the culture by plasmidless cells.
Degryse
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Department of Molecular Biology, Transgene S.A., Strasbourg, France.
D003001:Cloning, Molecular; D003960:Diaminopimelic Acid; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D007371:Interferon-gamma / Q000096:biosynthesis; D010957:Plasmids
D003960:Diaminopimelic Acid; D007371:Interferon-gamma