Question

a recombinant vector with a gene of interest inserted within a gene of alpha-galactosidase enzyme is introduced into a bacterium. explain the method that would help in the selection of recombinant colonies frm non recombinant ones? why this method of selection is reffered as Insertional Inactivation?

Answer 1

The selection of recombinants on the basis of antibiotic resistance genes as selectable markers is a cumbersome process because the cells have to be cultured in media containing different antibiotics and the recombinants have to be selected based on their ability or inability to grow in media containing the specific antibiotic. This can be overcome by using alternative markers. One of them is the gene coding for α-galactosidase. When foreign gene is inserted within α-galactosidase gene, the enzyme α-galactosidase gets inactivated (insertional inactivation).

The bacteria are then grown on a chromogenic substrate. The non-recombinants will produce blue-coloured colonies while the recombinants will produce colourless colonies because of the inactivation of the gene α-galactosidase.



Insertional inactivation is the loss of function of a gene such as Alpha-galactosidase gene in plasmid due to insertion of foreign DNA/transgene within it. This tool is used to select recombinants.