Question

You cloned an insert DNA into pBLU at the BamHI site and plated the transformants on the LA + ampicillin + X-gal plate to perform the blue/white screen. After overnight incubation at 37ºC, you observe a mixture of blue and white colonies. (Assume you have purified the insert-containing plasmid from chloramphenicol or kanamycin-resistant E. coli.)

Indicate the following:

-The molecular difference between the two populations/what is the genetic makeup of a white colony versus a blue colony

-Whether the results allow the inserted identity to be determined.

Answer 1

pBLU is a 5-5 kb plasmid containing AmpR gene, lacZ gene and a multiple cloning site in the lacZ gene.

In the above experiment, blue and white colonies were obtained. This is called blue white acreening.

Herein the white colonies are of recombinants while the blue ones are non recombinants..

The bacterial cells were grown on a LB-Amp-XGal plates.

Amp selects for transformants vs non transformants, since only those cells containing the plasmid (transformants) would be able to grow in the presence of Amp.

XGal encodes for the enzyme galactosidase which utilize the substarte XGal to produce an indigo coloured

Thus, those cells in which the insert has integrated at the BamHI site present within the lacZ gene (recombinants) would give white colonies since the gene is now disrupted by the insert.

On the other hand, non recombinants have a functional lacZ gene and so produce blue colonies.