Question

Some foreign DNA fragment is attached to Cla I site of pBR 322. This recombinant vector is used to transform Escherichia coli. Then on two different media, one containing ampicillin and the other containing tetracycline. The transformed cells containing the recombinant vector will

• A. Grow on both, tetracycline and ampicillin containing media
• B. Grow on ampicillin but not on tetracycline containing medium
• C. Grow on tetracycline but not on ampicillin containing medium
• D. Not grow on either tetracycline containing or ampicillin containing media

Answer 1

The correct option is A Grow on both, tetracycline and ampicillin containing media

Selectable marker
Selectable marker is a gene in a vector that helps in the selection of transformants (host cells which have successfully taken up the foreign DNA) and non-transformants (cells which have not taken up the vector DNA).
For example, antibiotic resistance genes in the vector.

pBR 322
pBR322 is the most common plasmid vector. It is named after Bolivar and Rodriguez.

Features of pBR 322
Common features of pBR322 are as follows:
● Restriction sites - It has multiple restriction sites for different restriction enzymes such as Pvu II, Pvu I, BamH I, EcoR I, Hind III, Pst I, Sal I, Cla I, etc.
● Rop - It is a specific base sequence that codes for the rop protein involved in the replication of the plasmid.
● Selectable markers - It has two selectable markers, one for ampicillin resistance ampR and one for tetracycline resistance tetR.
● Ori - It has an ori site or origin of replication site. Any piece of DNA when linked to this sequence can be made to replicate within the host cells.
● Pictorial representation of pBR322:

Role of ampR and tetR gene in pBR322
ampR and tetR gene present on pBR 322 provides ampicillin and tetracycline resistance to the host cell respectively. Therefore, host cells containing pBR322 can grow in nutrient media with ampicillin and tetracycline. These two genes act as selectable markers that are used to identify and differentiate transformed and non-transformed cells.

Effect of gene insertion on antibiotic resistance
A foreign gene or gene of interest inserted at restriction sites present on ampR (Pvu I and Pst I) or tetR (BamH I and Sal I) will inactivate the function of the antibiotic resistance genes respectively. Hence the transformed organism cannot grow on the medium containing the same antibiotic whereas non-transformants will grow in the medium.

Gene insertion at Cla I
The Cla I restriction site does not lie within the ampR and tetR gene. A foreign gene inserted at this site therefore will not inactivate the function of any of the antibiotic resistance genes. As a result the Escherichia coli cells that contain such a recombinant DNA will be able to grow in media containing ampicillin and tetracycline.

Final answer
(A) Grow on both, tetracycline and ampicillin containing media