BIOLOGY Volume 3 - A Guide to General Biology - 2004

25. APPLIED GENETICS

25.1. Bacterial Genetic Engineering

25.1.5. Stage 4. DNA Cloning

A single phage particle carrying a recombinant DNA molecule can produce over 10¹² identical copies of itself and the molecule in less than a day. E. coli Cells containing Plasmids are usually spread onto nutrient Agar in Petri dishes, where they divide every 30 minutes, eventually forming visible colonies. This yields just as many copies of the desired DNA as phage vectors do, with hundreds of plasmid copies synthesized within the dividing Bacteria as well. Both Methods allow billions of clones to be obtained in a short period of time. Before proceeding with further cloning, however, the transformed bacteria must be selected.

Selection of Transformed Bacteria

Let us consider a scenario where plasmids are used as vectors. When plasmid DNA is mixed with a bacterial culture, two problems arise. First, not all bacteria are transformed (take up plasmids). Second, not all plasmids carry the foreign donor DNA. These issues are cleverly bypassed by using plasmids that possess two special properties (Fig. 25.8).

1. The plasmids carry a Gene for resistance to a specific antibiotic. If this antibiotic is added to the nutrient medium used to culture the bacteria, only transformed cells (those containing plasmids) will be able to multiply and form colonies.

2. The plasmids carry a gene encoding the enzyme β-galactosidase, which also contains several restriction sites that do not disrupt the gene's function. β-Galactosidase is an enzyme that breaks down lactose into galactose and glucose. When this enzyme acts on the colorless substrate X-gal, it produces a blue-colored product. If foreign DNA is inserted into a restriction site within the gene, the gene becomes inactive. Therefore, if the bacteria that survived the antibiotic are subsequently grown on a medium containing X-gal, colonies lacking the donor DNA will turn blue. Conversely, bacteria forming colorless colonies contain the donor DNA and can be selected for subsequent cloning.

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Fig. 25.8. A plasmid vector carrying a gene for resistance to the antibiotic ampicillin. This gene allows us to select for plasmid-bearing cells on an ampicillin-supplemented medium, while cells lacking the plasmid will perish.