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The helper phage displaces a strand within thelZap which contains the foreign

DNA insert. This is circularised and packaged as a filamentous phage similar to M13

(Section 6.3.3). The packaged phagemid is secreted from theE. colicell and may be

recovered from the supernatant. Thus thelZap vector allows a number of diverse

manipulations to be undertaken without the necessity of recloning or subcloning

foreign DNA fragments. The process of subcloning is sometimes necessary when the

manipulation of a gene fragment cloned in a general purpose vector needs to be inserted

into a more specialised vector for the application of techniques such asin vitro

mutagenesis or protein production (Section 6.6).

6.3.3 M13 and phagemid-based vectors

Much use has been made of single-stranded bacteriophage vectors such as M13 and

vectors which have the combined properties of phage and plasmids, termed phage-

mids. M13 is a filamentous coliphage with a single-stranded circular DNA genome

(Fig. 6.20). Upon infection ofE. coli, the DNA replicates initially as a double-stranded

molecule but subsequently produces single-stranded virions for infection of further

bacterial cells (lytic growth). The nature of these vectors makes them ideal for

techniques such as chain termination sequencing (Section 6.6.1) andin vitromuta-

genesis (Section 6.6.3) since both require single-stranded DNA.

M13 or phagemids such as pBluescript SK infectE. coliharbouring a male-specific

structure termed the F-pilus (Fig. 6.21). They enter the cell by adsorption to this structure

and once inside the phage DNA is converted to a double-strandedreplicative formor

Antibiotic
resistance gene

M13mp

7.25 kb

ORI

ApR

lacZβ-galactosidase gene

Multiple cloning site

polylinker

Control regions for lacZ

Origin of replication

lacI

HindIII

SphI

PstI

HincII

AccI

SalI

BamHI

XmaI

SmaI

KpnI

SacI

EcoRI

Reverse sequencing primer

Forward sequencing primer

Fig. 6.20Genetic map and important features of bacteriophage vector M13.

216 Recombinant DNA and genetic analysis