The presence of chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert .presence of insert results into insertional inactivation of Beta galactosidase and recombinant colonies do not produce any colour these are identified as Recombinant Colony" explain these lines...

Dear student,

Non-recombinant bacteria form blue colonies due to the activation of the enzyme alpha galactosidase which hydrolyses alpha glyctosyl moiety from glycolipid and glycoproteins. But when recombinant bacteria is formed, the gene (GLA) encoding alpha galactosidase enzyme becomes inactivated due to insertion so white colony of bacteria is formed.

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.  


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