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 propaga te large, complex fragments of cloned DNA and are a core technology for fun ctional genomics. The simplest method of analyzing bacmid clones would invo lve a direct mutagenesis or allele exchange protocol utilizing positive and negative selectable markers, The utility of three different negative selec table markers to function in the context of a bacmid vector was therefore i nvestigated: sacB from Bacillus subtilis, which confers sensitivity to sucr ose; tetA from TN10, which confers resistance to tetracycline, osmotic sens itivity, and sensitivity to kanamycin and streptomycin; and rpsL from Esche richia coli, which confers sensitivity to streptomycin. When expressed indi vidually in the context of a bacmid vector, each of these markers confers a similar stringency of negative selection, with plating efficiencies on sel ective 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 stringen cy of selection of similar to 50- to similar to 1000-fold over that obtaine d with rpsL or tetA alone and similar to 20-fold more than that obtained us ing sacB, The combination of rpsL and tetA thus serves as the most efficien t positive and negative selectable marker system described to date. (C) 200 1 Academic Press.