ColE1 Vectors for Direct Selection of Cells Carrying a Hybrid Plasmid

Abstract

PKY2289, a ColE1∝ Tn3 derivative, useful for direct selection of cells carrying a hybrid plasmid, was deleted of its mobility functions and parts of the transposon including the left inverted repeat. This deletion mutant, named pKY2700 expresses low levels of colicin E1 synthesis even in recAcells. A nitrosoguanidine mutant of pKY2289 which shows a high level of constitutive colicin E1 synthesis was also deleted of the same sequences as pKY2700. The second plasmid, named pKY2800, has the same molecular weight (3.8 megadaltons) and almost the same structure as pKY2700, but produces colicin E1 at much higher levels and has a copy number 10 times higher. pKY2800 requires no colicin E1 induction for the direct selection of hybrid clones, while pKY2700 requires mitomycin C at a concentration of 10 ng per ml. These two ColE1 derivatives are useful as safe and convenient vectors for cloning DNA fragments at the cleavage sites of EcoRI, XmaI, and SstII of plasmid.