Cloning vector
Encyclopedia
A cloning vector is a small piece of DNA
into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with a restriction enzyme
that creates the same overhang, then ligating
the fragments together. There are many types of cloning vectors. Genetically engineered plasmid
s and bacteriophage
s (such as phage λ) are perhaps most commonly used for this purpose. Other types of cloning vectors include bacterial artificial chromosome
s (BACs) and yeast artificial chromosome
s (YACs).
so widespread.
In the case of expression vector
s, the main purpose of these vehicles is the controlled expression of a particular gene inside a convenient host organism (e.g. E. coli). Control of expression
can be very important; it is usually desirable to insert the target DNA
into a site that is under the control of a particular promoter. Some commonly used promoters are T7 promoters
, lac promoters
(bla promoter) and cauliflower mosaic virus's 35s promoter
(for plant vectors).
To allow for convenient and favorable insertions, most cloning vectors have had nearly all their restriction sites
engineered out of them and a synthetic multiple cloning site
(MCS) inserted that contains many restriction sites. MCSs allow for insertions of DNA into the vector to be targeted and possibly directed in a chosen orientation. A selectable marker, such as an antibiotic
resistance [e.g. beta-lactamase
(see figure)] is often carried by the vector to allow the selection of positively transformed
cells (see Screening below). All plasmids must carry a functional origin of replication
(ORI; not shown in figure).
Some other possible features present in cloning vectors are: vir genes
for plant transformation, integrase sites for chromosomal insertion, lacZα fragment
for α complementation and blue-white selection
, and/or reporter genes in frame with and flanking the MCS
to facilitate the production of recombinant proteins [e.g. fused to the Green fluorescent protein
(GFP) or to the glutathione S-transferase
(see figure)].
usually include a system for detecting the presence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used is the gene coding for E. coli β-galactosidase
, whose integrity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate X-gal
(5-bromo-4-chloro-3-indolyl-beta-d-galactoside) into an insoluble, blue product (5,5'-dibromo-4,4'-dichloro indigo). Cloning a fragment of DNA within the vector-based gene encoding the β-galactosidase prevents the production of an active enzyme. If X-gal is included in the selective agar plates, transformant colonies are generally blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA.
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with a restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
that creates the same overhang, then ligating
DNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
the fragments together. There are many types of cloning vectors. Genetically engineered plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
s and bacteriophage
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
s (such as phage λ) are perhaps most commonly used for this purpose. Other types of cloning vectors include bacterial artificial chromosome
Bacterial artificial chromosome
A bacterial artificial chromosome is a DNA construct, based on a functional fertility plasmid , used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell...
s (BACs) and yeast artificial chromosome
Yeast artificial chromosome
A yeast artificial chromosome is a vector used to clone DNA fragments larger than 100 kb and up to 3000 kb. YACs are useful for the physical mapping of complex genomes and for the cloning of large genes...
s (YACs).
Common features
Most commercial cloning vectors have key features that have made their use in molecular biologyMolecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
so widespread.
In the case of expression vector
Expression vector
An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation...
s, the main purpose of these vehicles is the controlled expression of a particular gene inside a convenient host organism (e.g. E. coli). Control of expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
can be very important; it is usually desirable to insert the target DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
into a site that is under the control of a particular promoter. Some commonly used promoters are T7 promoters
T7 phage
Bacteriophage T7 is a bacteriophage capable of infecting susceptible bacterial cells. It infects most strains of Escherichia coli Bacteriophage T7 is a bacteriophage capable of infecting susceptible bacterial cells. It infects most strains of Escherichia coli Bacteriophage T7 is a bacteriophage...
, lac promoters
Lac operon
The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. It consists of three adjacent structural genes, lacZ, lacY and lacA. The lac operon is regulated by several factors including the availability of glucose and of...
(bla promoter) and cauliflower mosaic virus's 35s promoter
Plant virus
Plant viruses are viruses that affect plants. Like all other viruses, plant viruses are obligate intracellular parasites that do not have the molecular machinery to replicate without a host. Plant viruses are pathogenic to higher plants...
(for plant vectors).
To allow for convenient and favorable insertions, most cloning vectors have had nearly all their restriction sites
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
engineered out of them and a synthetic multiple cloning site
Multiple cloning site
A multiple cloning site , also called a polylinker, is a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during...
(MCS) inserted that contains many restriction sites. MCSs allow for insertions of DNA into the vector to be targeted and possibly directed in a chosen orientation. A selectable marker, such as an antibiotic
Antibiotic
An antibacterial is a compound or substance that kills or slows down the growth of bacteria.The term is often used synonymously with the term antibiotic; today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic has come to denote a broader range of...
resistance [e.g. beta-lactamase
Beta-lactamase
Beta-lactamases are enzymes produced by some bacteria and are responsible for their resistance to beta-lactam antibiotics like penicillins, cephamycins, and carbapenems . These antibiotics have a common element in their molecular structure: a four-atom ring known as a beta-lactam...
(see figure)] is often carried by the vector to allow the selection of positively transformed
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
cells (see Screening below). All plasmids must carry a functional origin of replication
Origin of replication
The origin of replication is a particular sequence in a genome at which replication is initiated. This can either be DNA replication in living organisms such as prokaryotes and eukaryotes, or RNA replication in RNA viruses, such as double-stranded RNA viruses...
(ORI; not shown in figure).
Some other possible features present in cloning vectors are: vir genes
Agrobacterium
Agrobacterium is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus...
for plant transformation, integrase sites for chromosomal insertion, lacZα fragment
Lac operon
The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. It consists of three adjacent structural genes, lacZ, lacY and lacA. The lac operon is regulated by several factors including the availability of glucose and of...
for α complementation and blue-white selection
Blue white screen
The blue-white screen is a screening technique that allows for the detection of successful ligations in vector-based gene cloning. DNA of interest is ligated into a vector. The vector is then transformed into competent cell . The competent cells are grown in the presence of X-gal...
, and/or reporter genes in frame with and flanking the MCS
Multiple cloning site
A multiple cloning site , also called a polylinker, is a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during...
to facilitate the production of recombinant proteins [e.g. fused to the Green fluorescent protein
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
(GFP) or to the glutathione S-transferase
Glutathione S-transferase
Enzymes of the glutathione S-transferase family are composed of many cytosolic, mitochondrial, and microsomal proteins. GSTs are present in eukaryotes and in prokaryotes, where they catalyze a variety of reactions and accept endogenous and xenobiotic substrates.GSTs can constitute up to 10% of...
(see figure)].
Screening: example of the blue/white selection
Many general purpose vectors such as pUC19PUC19
pUC19 is one of a series of plasmid cloning vectors created by Messing and co-workers in the University of California. The p in its name stands for plasmid and UC represents the University in which it was created. It is a circular double stranded DNA and has 2686 base pairs...
usually include a system for detecting the presence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used is the gene coding for E. coli β-galactosidase
Beta-galactosidase
β-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins...
, whose integrity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate X-gal
X-gal
X-gal is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl...
(5-bromo-4-chloro-3-indolyl-beta-d-galactoside) into an insoluble, blue product (5,5'-dibromo-4,4'-dichloro indigo). Cloning a fragment of DNA within the vector-based gene encoding the β-galactosidase prevents the production of an active enzyme. If X-gal is included in the selective agar plates, transformant colonies are generally blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA.
See also
- Vector (molecular biology)Vector (molecular biology)In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes...
- Expression vectorExpression vectorAn expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation...
- IMAGE cDNA clonesIMAGE cDNA clonesIMAGE cDNA clones are a collection of DNA vectors containing cDNAs from various organisms including human, mouse, rat, non-human primates, zebrafish, pufferfish, Xenopus , and cow. Together they represent a more or less complete set of expressed genes from these organisms...
- phagemidPhagemidA phagemid or phasmid is a type of cloning vector developed as a hybrid of the filamentous phage M13 and plasmids to produce a vector that can grow as a plasmid, and also be packaged as single stranded DNA in viral particles...
- cosmidCosmidA cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid that contains cos sequences, DNA sequences originally from the Lambda phage. Cosmids can be used to build genomic libraries....
- fosmidFosmidFosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host can only contain one fosmid molecule. Fosmids are 40 kb of random genomic DNA...
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