What are the Common Methods Which Are Used Mainly For Selection of Recombinants in E. coli?

After the introduction of recombinant DNA into suitable host cells, it is essential to identify those cells which have received the recombinant DNA molecules. This process is called screening or selection. Selection of recombinant DNA cells is based on expression or non-expression of certain characters or traits.

The vector or foreign DNA present in recombinant cells express the characters, while the non-recombinants do not express the traits. Following are some of the methods which are used mainly for selection of recombinants in E. coli.

1. Direct Selection of Recombinants :

The above features ease the direct selection of recombinant cells. If the cloned DNA itself codes for resistance to the antibiotic ampicillin (amp^r) the recombinants can be allowed to grow on minimal medium containing ampicillin. Only such recombinants will grow and form colony on medium that contain amp^r gene on its plasmid vector.

Following this procedure you cannot say whether the recombinants growing on such medium contain re-ligated plasmid vector or contain recombinant plasmid plus foreign DNA fragment. Because amp^r gene is present in both the recombinants.

2. Insertional Selection Inactivation Method :

This is more efficient method than the direct selection. In this approach, one of the genetic traits is disrupted by inserting foreign DNA. Antibiotic resistance genes act as a good insertion inactivation system.

As discussed in earlier section that plasmid pBR322 contains two antibiotic resistance genes, one for ampicillin (amp^r gene) and the other for tetracycline (tet^r gene). If the target DNA is inserted into tef gene using BamH\, the property of resistance to tetracycline will be lost. Such recombinants would be test-sensitive.

When such recombinants (containing target DNA in tef gene) are grown onto medium containing tetracycline, they will not grow because their tef gene has been inactivated.

But they are resistant to ampicillin because amp^r gene is functional. On the other hand the self-ligated recombinants will show resistance to tetracycline and ampicillin. Therefore, they will grow on medium containing both the antibiotics.

3. Blue-White Selection Method :

It is also a powerful method used for screening of recombinant plasmid. In this method a reporter gene lacZ is inserted in the vector. The lacZ encodes the enzyme p-galactosidase and contains several recognition sites for restriction enzymes.

Beta-galactosidase breaks a synthetic substrate, X-gal (5-bromo-4-chloro-3- indolyl-P-D-galacto-pyranoside) into an insoluble blue coloured product. If a foreign gene is inserted into lacZ, this gene will be inactivated.

Therefore, no blue colour will develop. Because p-galactosidase is not synthesised due to inactivation of lacZ.

Therefore, the host cells containing rDNA will form white coloured colonies on the medium containing X-gal, whereas the other cells containing non- recombinant DNA will develop the blue coloured colonies. On the basis of colony colour the recombinants can be selected.