Phagemid vectors

[aayush_005]

Some times. generation of a single stranded DNA becomes important for DNA sequencing and site-directed mutagenesis. Using a single stranded DNA containing bacteriophage M13, a series of cloning vectors were developed. The bacteriophage f1 is closely related to M 13 which also infects E. coli. Phagemids are plasmids that contain origin of replication (ori) for single strand DNA of bacteriophage such as f1.
E. coli maintains a plasmid as double stranded DNA due to plasmid ori gene. If E. coli cells are infected by the helper fl phage, the ori of fl is activated. It switches to a mode of replication and produces single stranded DNA which is packed into phage particles as they are extruded from the host cell. Fig shows a phagemid pBlueScript II KS (+/-) which is used for generation of single stranded DNA molecules.
(a) pBlueScript II KS (+/-): This vector is 2961 bp long derivative from pUCI9. The 'KS' denotes the orientation of poly linker i.e. transcription of lacZ genes proceeds from the KpnI restriction site towards Sac1 restriction site. It consists of multiple cloning site (MCS) flanked by T3 and T7 promoters in opposite directions on the two strands. The MCS is set up for lacZ blue-white screening. A lac1 promoter complements with lacZ of E. coli upstream of lacZ region. This feature helps in
selection of recombinant vector based on the criteria of development of blue-white colonies (white colonies are formed if foreign DNA is inserted in the vector). It consists of fl (+) and fl (-) origin of replication obtained from a filamentous phage fl. It is used to get sense (+) and antisense (-) strands of lacZ gene after co-infecting the host with a helper phage. But in the absence of the helper phage an origin of replication (CoIEI ori from a plasmid) is used. Moreover, for antibiotic selection of recombinant phagemid vector, ampR (ampicillin resistance gene) is also used. In these genes a number of restriction sites are present in this vector.

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