1-4.2.1 Features of λ Phage Vectors:

The λ genome is linear, but in the ends has 12-nucleotides overhangs, termed as cos sites, which are complementary to each other. A λ cloning vector can be circularized using cos site which can be manipulated and replicated inside E. coli via the process of transfection.

Alternatively, a more efficient uptake system called in vitro packaging can be utilized. Treatment with the appropriate restriction endonuclease followed by ligation in presence of insert DNA, produces ‘ left arm-new DNA-right arm' concatemers that are then added to an in vitro packaging mix to form λ phage particles. These phages are then co-incubated with E. coli cells, and the infection process naturally transports the vector plus new DNA into the bacteria. Bacteria that are infected with the packaged cloning vector die within about 20 minutes and several rounds of phage replication and bacterial lysis forms a zone of clearing, called a plaque.

1-4.2.2 M13 Phage Vectors:

M13 phage is filamentous phage that infects E. coli via F-pilus . The genome is a single stranded circular DNA of size ~6.4kb surrounded by a proteinaceous coat. The DNA strand present in phage is called plus (+) strand. After entering to E. coli host, it converts into double stranded DNA molecule called replicative form (RF) by utilizing bacterial machinery. M13 phage as cloning vector can be obtained in both single stranded as well as double stranded form. Replicative form double stranded vector are modified and replicated inside E. coli host similar to a plasmid vector. Single stranded vectors can be isolated by collecting M13 phage. M13 vectors have useful application in following areas:

•  DNA sequencing

•  Mutagenesis study

•  probe generation

•  Phage display

Fig 1-4.2.2: M13 Phage Vectors