In insertional inactivation, the coding sequence of which enzyme is disrupted by the insertion of the recombinant DNA?

β-galactosidase

The method used to differentiate between recombinants and non-recombinants is insertional inactivation. In this technique, a selectable marker, such as an antibiotic resistance gene, is replaced by the foreign DNA (insert) within the coding sequence of an enzyme called β-galactosidase.

This insertion inactivates the gene responsible for the synthesis of β-galactosidase, resulting in the inability of the recombinant bacteria to produce this enzyme.

To identify recombinant colonies, a chromogenic substrate is added to the medium. Bacteria carrying non-recombinant plasmids (without the insert) will produce functional β-galactosidase and produce blue-colored colonies when exposed to the substrate. On the other hand, bacteria with recombinant plasmids (with the insert) will have the β-galactosidase gene inactivated, and these colonies will not produce any color. Thus, the lack of color indicates the presence of recombinant colonies. This method simplifies the identification of recombinant colonies without the need for simultaneous plating on two different antibiotic plates, making the process more efficient and less cumbersome.