Question

In a bacterial culture, some of the colonies produced blue colour in the presence of a chromogenic substrate and some did not due to the presence or absence of an insert (r DNA) in the coding sequence of (3 – galactosidase).

(a) Mention the mechanism and the steps involved in the above experiment.

(b) How is it advantageous over simultaneous plating on two plates having different antibiotics.

Answer 1

(a) The mechanism involved is called insertional inactivation, the phenomenon in which the enzyme becomes inactivated when a recombinant DNA is inserted with in the coding sequence of that enzyme.

Steps in process: 

• A recombinant DNA is inserted into the DNA sequence coding for the enzyme galacto sidase, it results in the inactivation of the enzyme.
• In case of the recombinants, when the plasmid has an insert, there is no blue colour produced in the presence of a chromogenic substrate in the medium.
• In the case of nonrecombinants/non- tranforments i.e. when the plasmid has no insert, a blue colour is produced in the presence of a chromogenic substrate in the medium.

(b) Advantage:

Simultaneous plating method is quite cumbersome as it requires two plates where as this method is simple and easy.