Question

Explain the method of Blue-White selection used for screening of recombinant cells containing desired plasmid with gene of interest.

Hint:

Blue-White screening uses lacZ disruption and X-gal substrate to distinguish recombinant (white) from non-recombinant (blue) bacterial colonies.

Answer 1

The Blue-White selection method is used to identify bacterial cells that have taken up recombinant plasmids containing the gene of interest. Principle: This technique relies on the disruption of the lacZ gene that encodes the enzyme β-galactosidase. The plasmid contains a multiple cloning site within the lacZ gene. When a foreign DNA fragment is inserted into this site, it disrupts lacZ function. Procedure:

• Bacterial cells are transformed with plasmids and grown on agar plates containing the antibiotic (to select for plasmid uptake) and X-gal, a chromogenic substrate for \(\beta\)-galactosidase.
• If the plasmid does not contain an insert (non-recombinant), \(\beta\)-galactosidase is active and cleaves X-gal, producing blue colonies.
• If the plasmid contains the insert disrupting lacZ (recombinant plasmid), \(\beta\)-galactosidase is inactive, and colonies remain white.

Result:

• Blue colonies: Non-recombinant cells without the gene of interest.
• White colonies: Recombinant cells containing the desired gene insert.

This method allows easy visual screening of recombinant bacterial colonies.