A '''phagemid''' or '''phasmid''' is a type of [[cloning]] [[Vector (biology)|vector]] developed as a hybrid of the [[M13 phage|M13(削除) ]] [[helper phage (削除ここまで)]] and [[plasmid]]s to produce a vector that can grow as a plasmid, and also be packaged as single stranded DNA in viral particles(削除) . One example of a phagemid is PRS314 which is of 4.78 [[Base_pair#Length_measurements|kb]] size (削除ここまで). Phagemids contain an [[Ori (genetics)|origin of replication (ori)]] for double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles. Many commonly used plasmids contain an f1 ori and are thus phagemids.
A '''phagemid''' or '''phasmid''' is a type of [[cloning]] [[Vector (biology)|vector]] developed as a hybrid of the [[M13 phage|M13]] and [[plasmid]]s to produce a vector that can grow as a plasmid, and also be packaged as single stranded DNA in viral particles. Phagemids contain an [[Ori (genetics)|origin of replication (ori)]] for double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles. Many commonly used plasmids contain an f1 ori and are thus phagemids(追記) . Similarly to a plasmid, a phagemid can be used to clone DNA fragments and be introduced into a bacterial host by a range of techniques (transformation, electroporation). However, infection of a bacterial host containing a phagemid with a 'helper' phage, for example VCSM13 or M13K07, provides the necessary viral components to enable single stranded DNA replication and packaging of the phagemid DNA into phage particles. These are secreted through the cell wall and released into the medium. Filamentous phage retard bacterial growth but, in contrast to lambda and T7 phage, are not generally lytic. Helper phage are usually engineered to package less efficiently that the phagemid so that the resultant phage particles contain predominantly phagemid DNA. Filamentous phage infection requires the presence of a [[pilus]] so only bacterial hosts containing the F' [[episome]] or its derivatives can be used to generate phage particles. Prior to the development of cycle sequencing phagemids were used to generate single stranded DNA template for sequencing purposes. Today phagemids are still useful for generating templates for [[site-directed mutagenesis]]. Detailed characterisation of the filamentous phage life cycle and structural features lead to the development of [[phage display]] technology, in which a range of peptides and proteins can be expressed as fusions to phage coat proteins and displayed on the viral surface. The displayed peptides and polypeptides are associated with the corresponding coding DNA within the phage particle and so this technique lends itself to a range of protein/protein and other ligand/receptor studies (追記ここまで).
A phagemid is different from a closely related cloning vector called a [[cosmid]]. While a cosmid can enter a host cell very effectively, almost all of the viral genes have been removed to make room for exogenous DNA, therefore the virus cannot replicate itself. In a phagemid, however, an origin of replication is present. Co-infection of a bacterial host containing a phagemid with a 'helper' virus such as f1, provides the necessary enzymes and proteins to enable single stranded DNA replication and packaging into phage particles. These are secreted through the cell wall and released into the medium. Filamentous phage retard bacterial growth but, in contrast to lambda and T7 phage, are not generally lytic. The helper virus is usually engineered to package less efficiently that the phagemid so that the resultant phage particles contain predominantly phagemid DNA.
One such phagemid, which is commonly used in molecular biology laboratories, is pBluescript II KS ,which is derived from pUC19 and is 2961 bp (base pair)long. The KS designation indicates the orientation of polylinker, such that the transcription of ''lac'' Z gene proceeds from the restriction site for ''Kpn''I to that for ''Sac''I
[[Category:Cloning]]
[[Category:Cloning]]
Revision as of 09:53, 13 March 2008
A phagemid or phasmid is a type of cloningvector developed as a hybrid of the M13 and plasmids to produce a vector that can grow as a plasmid, and also be packaged as single stranded DNA in viral particles. Phagemids contain an origin of replication (ori) for double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles. Many commonly used plasmids contain an f1 ori and are thus phagemids. Similarly to a plasmid, a phagemid can be used to clone DNA fragments and be introduced into a bacterial host by a range of techniques (transformation, electroporation). However, infection of a bacterial host containing a phagemid with a 'helper' phage, for example VCSM13 or M13K07, provides the necessary viral components to enable single stranded DNA replication and packaging of the phagemid DNA into phage particles. These are secreted through the cell wall and released into the medium. Filamentous phage retard bacterial growth but, in contrast to lambda and T7 phage, are not generally lytic. Helper phage are usually engineered to package less efficiently that the phagemid so that the resultant phage particles contain predominantly phagemid DNA. Filamentous phage infection requires the presence of a pilus so only bacterial hosts containing the F' episome or its derivatives can be used to generate phage particles. Prior to the development of cycle sequencing phagemids were used to generate single stranded DNA template for sequencing purposes. Today phagemids are still useful for generating templates for site-directed mutagenesis. Detailed characterisation of the filamentous phage life cycle and structural features lead to the development of phage display technology, in which a range of peptides and proteins can be expressed as fusions to phage coat proteins and displayed on the viral surface. The displayed peptides and polypeptides are associated with the corresponding coding DNA within the phage particle and so this technique lends itself to a range of protein/protein and other ligand/receptor studies.
