Much use has been made of single-stranded bacteriophage vectors such as fd, M13 and vectors that have the combined properties of phage and plasmids, termed phagemids. M13 is a filamentous coliphage with a single-stranded circular DNA genome (Figure 1). Upon infection of E. 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 and in vitro mutagenesis, since both require single-stranded DNA.

Fig1. Genetic map and important features of bacteriophage vector M13.

M13 or phagemids such as pBluescript SK infect E. coli harbouring a male-specific structure termed the F-pilus (Figure 2). They enter the cell by adsorption to this structure and once inside, the phage DNA is converted to a double-stranded replicative form or RF DNA. Replication then proceeds rapidly until some 100 RF molecules are produced within the E. coli cell. 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 the E. coli cell 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.

Fig2. Life cycle of bacteriophage M13. The bacteriophage virus enters the E. coli cell 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 the E. coli cell membrane.

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 infected E. coli cells and purifying the supercoiled circular phage DNA with the same methods used for plasmid isolation. Intact single-stranded DNA packaged in the phage protein coat located in the super natant may be precipitated with reagents such as polyethylene glycol, and the DNA purified with phenol/chloroform. 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, including M13mp8/9, mp18/19, etc., all of which have a number of highly useful features. They all contain a synthetic MCS, which is located in the lacZ gene without disruption of the reading frame of the gene. This allows efficient selection to be undertaken based on the technique of blue/white screening. As the series of vectors were developed, the number of restriction sites was increased in an asymmetric fashion. Thus M13mp8, mp12, mp18 and sister vectors that have the same MCS but in reverse orientation, M13mp9, mp13 and mp19, respectively, have more restriction sites in the MCS making the vector more useful since a greater choice of restriction enzymes is available (Figure 3). However, one problem frequently encountered with M13 is the instability and spontaneous loss of inserts that are greater than 6 kb.

Fig3. Design and orientation of polylinkers in M13 series. Only the main restriction enzymes are indicated.

Phagemids are very similar to M13 and replicate in a similar fashion. One of the f irst phagemid vectors, pEMBL, was constructed by inserting a fragment of another phage termed f1, containing a phage origin of replication and elements for its morphogenesis, into a pUC8 plasmid. Following superinfection with helper phage, the f1 origin is activated allowing single-stranded DNA to be produced. The phage is assembled into a phage coat extruded through the periplasm and secreted into the culture medium in a similar way to M13. Without superinfection, the phagemid replicates as a pUC-type plasmid and in the replicative form (RF) the DNA isolated is double-stranded. This allows further manipulations such as restriction digestion, ligation and mapping analysis to be performed. The pBluescript SK vector is also a phagemid and can be used in its own right as a cloning vector and manipulated as if it were a plasmid. It may, like M13, be used in nucleotide sequencing and site-directed muta genesis, and it is also possible to produce RNA transcripts that may be used in the production of labelled cRNA probes or riboprobes.

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ابتكار من جامعة الشارقة منخفض التكلفة للحماية من الزلازل

اكتشاف تأثير غير متوقع للكاكاو على وظائف الدماغ

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مواضيع ذات صلة

Cloning Vectors : Cosmid-Based Vectors

Cloning Vectors : Virus-Based Vectors

Cloning Vectors : Plasmids

The TATA Box, Initiators, and CpG Islands Function as Promoters in Eukaryotic DNA

Chromosome Painting and DNA Sequencing Reveal the Evolution of Chromosomes

During Metaphase, Chromosomes Can Be Distinguished by Banding Patterns and Chromosome Painting

Chromosome Number, Size, and Shape at Metaphase Are Species-Specific

Origins and Types of Mutations

Regulation of Gene Expression

The Human Genome

The Polymerase Chain Reaction (PCR)

Additional Nonhistone Proteins Regulate Transcription and Replication