Sumo15A: a lambda phasmid that permits easy selection for and against cloned inserts.
We report the construction of a phasmid vector, Sumo15A, designed for recombination-based screening of recombinant DNA libraries [Seed, Nucleic Acids Res. 11 (1983) 2427-2445]. This vector permits rapid selection in Escherichia coli for homology-mediated integration and excision between homologous DNA inserts cloned in a supF-carrying plasmid and in Sumo15A. The region available for recombination spans the homologous sequence shared by the plasmid and the phasmid. SupF is the selection tool that we used. Efficient selection for supF expression by Sumo15A requires recombination mediated by the lambda phage red gene, which promotes homologous recombination between phage and plasmid DNAs. Counterselection against supF expression by Sumo15A occurs because the presence of a pSC101-derived plasmid replicon in this phasmid permits the growth of Sumo15A as a plasmid in a specialized host, E. coli strain DK37. In strain DK37, Sumo15A cannot replicate as a phage, and the presence of a plasmid-carrying supF is lethal to cells plated on galactose plates. This scheme was developed to select for sequences that are transcribed from chromosomes of interest.
Kurachi
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Department of Pediatrics, Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor 48109-0650.
Baldori
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N
Kurnit
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DM
D010582:Bacteriophage lambda / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D005822:Genetic Vectors; D010957:Plasmids; D012093:Replicon
D004251:DNA Transposable Elements