at allow a gene to be conveniently inserted into the vector or removed from it. This may be a multiple cloning site (MCS), which contains many unique restriction sites. The restriction sites in the MCS are first cleaved by restriction enzymes, and a PCR-amplified target gene, also digested with the same enzymes, is then ligated into the vectors using DNA ligase. The target DNA sequence can be inserted into the vector in a specific direction if so desired. The restriction sites may be further used for sub-cloning into another vector if necessary.
Other cloning vectors may use topoisomerase instead of ligase and cloning may be done more rapidly without the need for restriction digest of the vector or insert. In this TOPO cloning method a linearized vector is activated by attaching topoisomerase I to its ends, and this "TOPO-activated" vector may then accept a PCR product by ligating both the 5' ends of the PCR product, releasing the topoisomerase and forming a circular vector in the process. Another method of cloning without the use of DNA digest and ligase is by DNA recombination, for example as used in the Gateway cloning system. The gene, once cloned into the cloning vector (called entry clone in this method), may be conveniently introduced into a variety of expression vectors by recombination.
A selectable marker is carried by the vector to allow the selection of positively transformed cells. Antibiotic resistance is often used as marker, an example being the beta-lactamase gene, which confers resistance to the penicillin group of beta-lactam antibiotics like ampicillin. Some vectors contain two selectable markers, for example the plasmid pACYC177 has both ampicillin and kanamycin resistance gene. Shuttle vector which is designed to be maintained in two different organisms may also require two selectable markers, although some selectable markers such as resistance to zeocin and hygromycin B are effective in different cell types. Auxotrophic selection markers that allow an auxotrophic organism to grow in minimal growth medium may also be used; examples of these are LEU2 and URA3 which are used with their corresponding auxotrophic strains of yeast.
Another kind of selectable marker allows for the positive selection of plasmid with cloned gene. This may involve the use of a gene lethal to the host cells, such as barnase, Ccda, and the parD/parE toxins. This typically works by disrupting or removing the lethal gene during the cloning process, and unsuccessful clones where the lethal gene still remains intact would kill the host cells, therefore only successful clones are selected.
Reporter genes are used in some cloning vectors to facilitate the screening of successful clones by using features of these genes that allow successful clone to be easily identified. 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.
Elements for expression
A cloning vector need not contain suitable elements for the expression of a cloned target gene, such as a promoter and ribosomal binding site (RBS), many however do, and may then work as an expression vector. The target DNA may be inserted into a site that is under the control of a particular promoter necessary for the expression of the target gene in the chosen host. 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 commonly used promoters are the T7 and lac promoters. The presence of a promoter is necessary when screening techniques such as blue-white selection are used.
Cloning vectors without promoter and RBS for the cloned DNA sequence are sometimes used, for example when cloning genes whose products are toxic to E. coli cells. Promoter and RBS for the cloned DNA sequence are also unnece