Question

If a person obtains transformants by inserting a recombinant DNA within the coding sequence of enzyme-galactosidase, he will separate out recombinants from non- recombinants by which of the following observations?
(a) Non- recombinant colonies do not produce any color whereas recombinants give blue- colored colonies
(b) Recombinant colonies do not produce any color whereas non- recombinants give blue- colored colonies
(c) Recombinants and non-recombinants both produce blue colored colonies
(d) No colonies are formed due to insertional inactivation

Answer 1

A recombinant DNA is the one where there are two different sources of DNA in a single genome. The enzyme galactosidase can hydrolyze a substrate to release a colored product. It is a type of selective marker used by biologists to separate out recombinants from non- recombinants.

Complete answer:
The recombinants can be separated and differentiated from the colony of recombinants by blue- white screening. This type of screening takes the advantage of a $\beta$-galactosidase enzyme that is naturally produced by E.coli bacteria. It is a part of the lac operon and is also a selective marker. The recombinants do not secrete $\beta$-galactosidase and thus no color is produced whereas non- recombinants give blue- colored colonies.
So, the answer is ‘Recombinant colonies do not produce any color whereas non-recombinants give blue- colored colonies .’

Additional Information:
- In recombination, a segment of desired foreign DNA is inserted into the coding sequence of the $\beta$-galactosidase enzyme in the genome of E.coli with the help of restriction enzymes.
- The insertion of foreign DNA in the coding sequence of the enzyme would eventually render the recombinants inactive. This phenomenon is known as insertional inactivation.
- For the selection of recombinant clones from the recombinant ones, a chromogenic substance is added in the medium. This chromogenic substance is named X-gal.
- The enzyme $\beta$-galactosidase converts X-gal into an insoluble blue-colored product. Thus, the non- recombinant clones will be able to form a blue- colored colony due to the efficient production of the $\beta$-galactosidase enzyme as its sequence was unaltered due to the non-addition of foreign DNA.
- This will not be the case of recombinant colonies and they will appear white. This is due to their inefficient production of the $\beta$-galactosidase enzyme as its sequence has been inactivated by the insertion of the desired foreign DNA to be cloned.

Note: An operon is a group of genes with a single promoter for all three of them. Such a type of arrangement of genes is a characteristic feature of the prokaryotic transcriptional unit. If the genes in this operon are specifically coding for the enzymes that will eventually break down the lactose, then such type of operon is known as lac operon. This operon is functionally active in the presence of lactose.