The colonies of recombinant bacteria appear white in contrast to blue colonies of non­ recombinant bacteria because of?

Explanation

The presence of restriction sites within the markers tet r and amp r of plasmid permits an easy selection for cells transformed with recombinant plasmid. Insertion of the DNA fragment into the plasmid makes antibiotic resistance genes nonfunctional, for example, insertion of the DNA fragment into the plasmid (pBR322) using Pst I or Pvu I makes amp r nonfunctional. Bacterial cells containing such a recombinant pBR322 will be unable to grow in the presence of ampicillin but will grow on tetracycline. This process, however, becomes burdensome because it requires simultaneous plating on two plates having different antibiotics. Thus, an alternative selectable marker is developed to differentiate recombinants and non­ recombinants on the basis of their ability to produce colour in the presence of a chromogenic substance. Here, a recombinant DNA is inserted in the coding sequence of an enzyme B­-galactosidase. pUC 18 plasmid contains this gene which allows it to produce B-galactosidase which degrades certain sugars and produces a blue pigment when exposed to a specific substrate analog. If the plasmid in the bacterium does not have an insert, i.e., is non­ recombinant, the presence of chromogenic substrate gives blue coloured colonies. Presence of insert in the plasmid in recombinant bacterium does not produce any colour, such bacterial colonies are marked as recombinant colonies.