Cloning vector

  A cloning vector is a small DNA vehicle that carries a foreign DNA fragment. 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. 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).

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Common Features

Most commercial cloning vectors have a couple of key features that have made their use in molecular biology so widespread.

Usually, the main purpose of cloning vector is the controlled expression of a particular gene inside a convenient host organism (eg. 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 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 antibiotic resistance [eg. beta-lactamase (see figure)] is often included in the vector to identify positively transformed cells. All inserted DNA (plasmids etc.) need an origin of replication (ORI; not shown in figure). High stringency ORIs are preferable for cloning vectors.

Some other possible features of cloning vectors are: vir genes for plant transformation, intergrase sites for chromosomal insertion, lac Z alpha fragment for blue-white selection, and/or in-frame genes attached to the MCS for recombinant proteins [eg. Green fluorescent protein (GFP) or glutathione S-transferase (see figure)].

Screening: blue and white selection

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 beta-galactosidase can be detected by its ability to change the substrate X-gal (5 bromo-4-chloro-3-indolyl-beta-d-galactoside) from colourless to blue. Cloning a fragment of DNA within the vector based gene encoding the beta galactosidase prevents the formation of an active beta galactosidase. If x gal is included in the selective agar plates, transformant colonies are 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.

References

B. R. Glick and J. J. Pasternak (2005). Molecular Biotechnology Principles and Applications of Recombinant DNA. 3rd ed. ASM Press Washington, D. C.