A plasmid cloning system utilizing replication and packaging functions of the filamentous bacteriophage fd

Abstract

DNA cloning vectors were developed which utilize the replication origin (ori) of bacteriophage fd for their propagation. These vectors depend on the expression of viral gene 2 that was inserted into phage lambda, which in turn was integrated into the host genome. The constitutive expression of gene 2 in the host cells is sufficient for the propagation of at least 100 pfd plasmids per cell. In addition to the fd ori, the pfd vectors carry various antibiotic-resistance genes and unique restriction sites. Some of these vectors have no homologies to commonly used pBR plasmids or to lambda DNA. The nucleotide sequence of the vectors can be deduced from published sequences. Large DNA inserts can be stably propagated in pfd vectors; these are more stable than similar DNA fragments cloned in intact genomes of filamentous bacteriophage. Inclusion of phage sequences required for efficient phage packaging and infection with a helper phage resulted in formation of phage particles containing single-stranded plasmid genomes. Growth at 42 degrees C without selective pressure results in loss of pfd plasmids.