An improved bacteriophage lambda vector: construction of model recombinants coding for kanamycin resistance

Abstract

An attenuated bacteriophage lambda has been prepared for proposed use as an EK2 vector. This phage, designated lambdagt vir Jam27 Zam718-lambdaB' can accomodate up to 11.10(6) daltons of foreign DNA inserted through Eco RI ends. The virulence mutations and nin 5 reduce the frequency of lysogen and/or plasmid formation. The mutations Jam27 and Zam718 require a suppressor in the bacterial host. The phage recombination functions contained in the EcoRIlambdaC fragment have been deleted, and only the EcoRIlambdaB fragment remains (in reverse orientation) in the center portion of the vector. In addition, this phage adsorbs to sensitive bacteria at a significantly reduced rate, conferring another block to the escape of free phage. Model recombinants have been constructed by in vitro recombination with an EcoRI fragment coding for kanamycin resistance (originally derived from R-factor R6-5). This fragment of DNA is 4.6.10(6) daltons in size, contains an inverted repeat, and also appears to contain a promoter for the kanamycin resistance gene. Using this model recombinant, the rate of transfer of kanamycin resistance to permissive and nonpermissive strains of E. coli has been measured.