Synergy between tetA and rpsL provides high-stringency positive and negative selection in bacterial artificial chromosome vectors.
Bacterial artificial chromosome (bacmid) vectors are used to stably propagate large, complex fragments of cloned DNA and are a core technology for functional genomics. The simplest method of analyzing bacmid clones would involve a direct mutagenesis or allele exchange protocol utilizing positive and negative selectable markers. The utility of three different negative selectable markers to function in the context of a bacmid vector was therefore investigated: sacB from Bacillus subtilis, which confers sensitivity to sucrose; tetA from TN10, which confers resistance to tetracycline, osmotic sensitivity, and sensitivity to kanamycin and streptomycin; and rpsL from Escherichia coli, which confers sensitivity to streptomycin. When expressed individually in the context of a bacmid vector, each of these markers confers a similar stringency of negative selection, with plating efficiencies on selective media of 2.3 x 10(-5), 9.4 x 10(-4), and 5.7 x 10(-5), respectively. However coexpression of rpsL and tetA results in a synergistic enhancement of the osmotic, kanamycin, and streptomycin sensitivities, with a stringency of selection of approximately 50- to approximately 1000-fold over that obtained with rpsL or tetA alone and approximately 20-fold more than that obtained using sacB. The combination of rpsL and tetA thus serves as the most efficient positive and negative selectable marker system described to date.
Stavropoulos
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TA
Gene Therapy and Molecular Virology Group, The John P. Robarts Research Institute, London, Ontario, N6A 5K8, Canada.
Strathdee
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CA
D017920:Antiporters / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005819:Genetic Markers; D005822:Genetic Vectors; D006602:Hexosyltransferases / Q000235:genetics; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics*
D017920:Antiporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005819:Genetic Markers; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C000717190:RpsI protein, E coli; C110201:ScoS9 protein, Streptomyces coelicolor; C079051:tetA protein, Bacteria; D006602:Hexosyltransferases; C021513:levansucrase