Plasmid transformation
In order to persist and be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the chromosome. Because transformation usually produces a mixture of rare transformed cells and abundant non-transformed cells, a method is needed to identify the cells that have acquired the plasmid. Plasmids used in transformation experiments will usually also contain a gene giving resistance to an antibiotic that the intended recipient strain of bacteria is sensitive to. Cells able to grow on media containing this antibiotic will have been transformed by the plasmid, as cells lacking the plasmid will be unable to grow.
Another marker, used for identifying E. coli cells that have acquired recombinant plasmids, is the lacZ gene, which codes for β-galactosidase. Because β-galactosidase is a homo-tetramer, with each monomer made up of one lacZ-α and one lacZ-ω protein, if only one of the two requisite proteins is expressed in the resulting cell, no functional enzyme will be formed. Thus, if a strain of E. coli without lacZ-α in its genome is transformed using a plasmid containing the missing gene fragment, transformed cells will produce β-galactosidase, while untransformed cells will not, as they are only able to produce the omega half of the monomer. In this type of transformation, the polylinker region of the plasmid lies in the lacZ-α gene fragment, meaning that successfully produced recombinant plasmids will have the desired gene inserted somewhere within lacZ-α. When this disrupted gene fragment is expressed by E. coli, no usable lacZ-α protein is produced, and therefore no usable β-galactosidase is formed. When grown on media containing the colorless, modified galactose sugar X-gal, colonies that are able to metabolize the substrate (and that have therefore been transformed, but not by recombinant plasmids) will appear blue in color; colonies that are not able to metabolize the substrate (and that have therefore been transformed by recombinant plasmids) will appear white.
