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 similar to 50- to similar to 1000-fold over that obtained with rpsL or tetA alone and similar to 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. (C) 2001 Academic Press.