Question

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

• A. Non-recombinant bacteria containing $\beta$-galactosidase.
• B. Insertional inactivation of $\alpha$-galactosidase in non-recombinant bacteria.
• C. Insertional inactivation of $\alpha$-galactosidase in recombinant bacteria.
• D. Inactivation of galactase enzyme in recombinant bacteria.

Answer 1

Answer: (C)

Insertional inactivation of $\alpha$-galactosidase in recombinant bacteria.

• Blue-white screening allows rapid detecting of the recombinant bacteria in vector-based cloning experiments.
• The blue-white screening method works by disrupting the $\alpha$-complementation process.
• The plasmid carries within the lacZ $\alpha$ sequence an internal multiple cloning site or MCS.
• This MCS can be cut by restriction enzymes so that the foreign DNA may be inserted, thereby disrupting the gene and production of $\alpha$-peptide.
• Consequently, in cells containing the plasmid with an insert, no functional $\beta$-galactosidase may be formed. The presence of an active $\beta$-galactosidase can be detected by X-gal, a colourless analogue of lactose, that may be cleaved to form a bright blue insoluble pigment.
• This results in a characteristic blue colour in cells containing the functional $\beta$-galactosidase.
• Blue colonies, therefore, show that they may contain a vector, while white colonies indicate the presence of an insert in lacZ $\alpha$ which disrupts the formation of an active $\beta$-galactosidase.

Thus, the correct answer is option C.