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RF DNA. Replication then proceeds rapidly until some 100 RF molecules are produced

within theE. colicell. DNA synthesis then switches to the production of single strands

and the DNA is assembled and packaged into the capsid at the bacterial periplasm. The

bacteriophage DNA is then encapsulated by the major coat protein, gene VIII protein,

of which there are approximately 2800 copies with three to six copies of the gene III

protein at one end of the particle. The extrusion of the bacteriophage through the

bacterial periplasm results in a decreased growth rate of theE. colicell rather than host

cell lysis and is visible on a bacterial lawn as an area of clearing. Approximately 1000

packaged phage particles may be released into the medium in one cell division.

In addition to producing single-stranded DNA the coliphage vectors have a number

of other features that make them attractive as cloning vectors. Since the bacteriophage

DNA is replicated as a double-stranded RF DNA intermediate a number of regular DNA

manipulations may be performed such as restriction digestion, mapping and DNA

ligation. RF DNA is prepared by lysing infectedE. colicells and purifying the super-

coiled circular phage DNA with the same methods used for plasmid isolation. Intact

single-stranded DNA packaged in the phage protein coat located in the supernatant may

be precipitated with reagents such as polyethylene glycol, and the DNA purified with

phenol/chloroform (Section 5.7.1). Thus the bacteriophage may act as a plasmid under

certain circumstances and at other times produce DNA in the fashion of a virus.

A family of vectors derived from M13 are currently widely used termed M13mp8/9,

M13 phage released into medium

without lysing E. coli cells

M13 adsorbs to

E. coli through

F-pilus

Single-stranded DNA is

assembled at periplasm

RF

Rolling circle

replication

++ + +

strand

Single-stranded

DNA enters

E. coli cell

Fig. 6.21Life cycle of bacteriophage M13. The bacteriophage virus enters theE. colicell through the F-pilus. It

then enters a stage where the circular single strands are converted to double strands. Rolling-circle replication

then produces single strands, which are packaged and extruded through theE. colicell membrane.

217 6.3 Cloning vectors