Cloning vector

[1] The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium.

Thus, the cloning vectors used often have elements necessary for their propagation and maintenance in E. coli, such as a functional origin of replication (ori).

Some vectors contain two selectable markers, for example the plasmid pACYC177 has both ampicillin and kanamycin resistance gene.

Such features present in cloning vectors may be the lacZα fragment for α complementation in blue-white selection, and/or marker gene or reporter genes in frame with and flanking the MCS to facilitate the production of fusion proteins.

Examples of fusion partners that may be used for screening are the green fluorescent protein (GFP) and luciferase.

Where the promoter is present, the expression of the gene is preferably tightly controlled and inducible so that proteins are only produced when required.

Some vectors are designed for transcription only with no heterologous protein expressed, for example for in vitro mRNA production.

However low-copy-number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells.

In replacement vectors, the cleavage sites flank a region containing genes not essential for the lytic cycle, and this region may be deleted and replaced by the DNA insert in the cloning process, and a larger sized DNA of 8–24 kb may be inserted.

Yeast artificial chromosome are used as vectors to clone DNA fragments of more than 1 mega base (1Mb=1000kb) in size.

These vectors also contain suitable restriction sites to clone foreign DNA as well as genes to be used as selectable markers.

It can carry very large DNA fragment (there is no upper limit on size for practical purposes), therefore it does not have the problem of limited cloning capacity of other vectors, and it also avoids possible insertional mutagenesis caused by integration into host chromosomes by viral vector.

A vector based on Simian virus 40 (SV40) was used in first cloning experiment involving mammalian cells.

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 activity 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 lacZα sequence of 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.

Schematic representation of the pBR322 plasmid, one of the first plasmids widely used as a cloning vector.
The pUC plasmid has a high copy number, contains a multiple cloning site (polylinker), a gene for ampicillin antibiotic selection, and can be used for blue-white screen.
An LB agar plate showing the result of a blue white screen. White colonies may contain an insert in the plasmid it carries, while the blue ones are unsuccessful clones.