Cloning Vectors (Vehicle DNA)

Cloning vectors, or vehicle DNA, are DNA molecules that carry a foreign DNA segment and replicate it inside a host cell. Vectors can be plasmids, bacteriophages (such as M13 or lambda phage), cosmids, phagemids, BACs (bacterial artificial chromosomes), YACs (yeast artificial chromosomes), transposons, baculoviruses, or mammalian artificial chromosomes (MACs); of these, plasmid vectors (pBR322, pUC, the Ti plasmid) and bacteriophages (lambda, M13) are the ones most commonly used.

A good vector needs two key properties: the ability to replicate independently of the host chromosome (through its own ori/origin-of-replication gene), so that as it replicates, many copies of the inserted DNA are produced too; and the ability to get into host cells easily. Beyond this, a useful cloning vector also needs a marker gene for antibiotic resistance (so transformed cells can be picked out later), a unique cleavage site for the chosen restriction enzyme inside one of its marker genes, and control elements such as a promoter, an operator and ribosome-binding sites so the inserted gene can actually be expressed. Naturally occurring plasmids do not have all of these features built in, so they are engineered to have them -- pBR322, pBR320 and pACYC177 are examples of such constructed plasmids, and pBR322 is the plasmid most commonly used in plant rDNA technology (Fig 12.4 shows a typical plasmid map with its ori, ampr marker and polylinker/multiple-cloning-site region).

For carrying new DNA into plants specifically, an important natural vector is the Ti (tumour-inducing) plasmid found in Agrobacterium tumefaciens, a soil bacterium that causes the plant disease crown gall (marked by tumour-like overgrowths on the plant). The Ti plasmid carries a transposon called T-DNA, which inserts copies of itself into the chromosomes of infected plant cells; a plant cell carrying this inserted DNA can then be grown in culture or induced to develop into a whole new transgenic plant. …