Everything about Cloning Vector totally explained
A
cloning vector is a small
DNA vehicle 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 the same
restriction enzyme, then
ligating the fragments together. There are many types of cloning vectors. Genetically engineered
plasmids and
bacteriophages (such as phage λ) are perhaps most commonly used for this purpose. Other types of cloning vectors include
bacterial artificial chromosomes (BACs) and
yeast artificial chromosomes (YACs).
Common Features
Most commercial cloning vectors have key features that have made their use in
molecular biology so widespread.
In the case of
expression vectors, the main purpose of these vehicles is the controlled expression of a particular gene inside a convenient host organism (eg.
E. coli). Control of
expression can be very important; it's usually desirable to insert the target
DNA into a site that's 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 seleop;jnopctable marker, such as an
antibiotic resistance [eg.
beta-lactamase (see figure)] is often carried by the vector to allow the selection of positively
transformed cells (see 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,
intergrase 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 [eg.fused to the
Green fluorescent protein (GFP) or to the
glutathione S-transferase (see figure)].
Screening: example of the blue/white selection
Many general purpose vectors such as pUC19 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.
Further Information
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