The Recombinant Vector is constructed in vitro it is then generally introduced into E.coli (1) select the recombinant from the unchanged vector, (2) to obtain many copies of the recombinant vector or the DNA insert, or (3) to express the insert in E.coli itself, purified recombinant vector may subsequently be introduced into another bacterium, e.g Bacillus subtitilis, streptomyces etc. yeast, higher plants or animals. The various approaches for introducing recombinant vectors briefly described while considering the various types of vectors.
Increased competence of E. coli by CaCl2 treatment. E.coli cells are generally poorly accessible to DNA molecules. But treatment with CaCl2 makes them permeable to DNA the process involved is poorly understood growing E.coli cells are isolated and suspended in 50mM cacl2 at a concentration of 10*8 cells the cells may be incubated for 12-24 hours to increase the frequency of transformation. The recombinant vector is then added efficient transformation takes transformed clones. The frequency of transformed cells is per mg of plasmid DNA this is about one transformation per 10,000 plasmid molecules. This frequency can be further improved by using special E.coli strains e.g. SK 1590, SK 1592, etc.
Infection by vectors packaged as virions. Alternatively, those vectors that have the phage cos sequences e.g cosmids, phasmids and vectors, are generally packaged in vitro into specially produced empty phage haeads and complete particles are constituted. These phage particles are used to infect E.coli cells. These vectors can also be used to transform E.coli cells directly as naked DNA, using the CaCl2 technique. Generally infection by phage particles containing DNA insert is far more efficient than direct transformation. For example, the frequency of infection by recombinant phage vectors packaged in phage particles is up to 10*8 plaques of DNA while it is less than 10*3 DNA when the recombinant vector is used for transformation by the CaCl2 technique. The infected transformed bacterial cells are spread on lawn of susceptible cells. Where celar areas or plaques develop in the lawn. Plaques containing the recombinant vector identified and the phage particles collected from such plaques provide the purified vector.