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Home >> Recombinant DNA Technology >> Introduction of the Recombinant DNA Into the Suitable Host
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Introduction of the Recombinant DNA Into the Suitable Host - The recombinant DNA is constructed in vitro; it is then generally introduced into E. coli to
(1) select the recombinant DNAs from the unchanged vector,
(2) to obtain many copies of the recombinant DNA, or
(3) to express the DNA insert in E. coli itself. Purified recombinant DNA may subsequently be introduced into another bacterium, e.g. Bacillus subtilis, Streptomyces, etc., yeast, higher plants or animals. The various approaches for introducing recombinant DNAs into bacteria, especially E. coli, are briefly summarised here.

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 50 mM CaCl2 at a concentration of 108-1010 cells/ml; the cells may be incubated for 12- 24 hr to increase the frequency of transformation.

The recombinant DNA is then added; efficient transformation takes only a few minutes and the cells are plated on a suitable medium for the selection of transformed clones. The frequency of transformed cells is 106-107 per μg 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., SK1590, SK1592, X1766, etc., and some specific conditions during transformation; these may raise the frequency to 5 X 108 transformed cells/μg of plasmid DNA.

The transformed cells are suitably diluted and spread thinly on a suitable medium so that each cell is well separated and produces a separate colony. Generally, the medium is so designed also permit only the transformed cells to divide and produce colonies.

Infection by Recombinant DNAs Packaged as Virions Alternatively, those recombinant DNA that have the λ phage cas sequences, e.g., those derived from cosmids, phasmids and λ vectors, are generally packaged in vitro into specially produced empty λ phage heads and complete A particles are constituted.

These phage particles are used to infect E. coli cells; this process is often called transfection. These recombinant DNAs can also be used to transform E. coli cells directly as naked DNA, using the CaCl2 technique. Generally, transfection is far more efficient than direct transformation.

For example, the frequency of transfection by recombinant λ phage DNAs packaged in phage particles is up to 108 plaques/μg of DNA, while it is less than 103 plaques/μg DNA when the recombinant DNA is used for transformation by the CaCl2 technique.

The infected/transformed bacterial cells are spread on a lawn of susceptible cells, where clear areas or plaques develop in the lawn. Plaques containing the recombinant DNA (A vector and phasmids) are identified and the phage particles collected from such plaques provide the purified vector/ recombinant DNA.
 
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