Introduction- The different vectors are used in cloning techniques to produce recombinant DNA or clone. Transformation of recombinant DNA into the suitable host gives colonies and screening of the clone containing desired gene fragment is required for down-stream applications.

Chromogenic Substrate- The use of chromogenic substrate to detect a particular enzymatic activity is the basis to screen the desired clone. The most popular system to exploit this feature is “Blue white screening” where a coloroless substrate is processed to a colored compound. The colorless compound X-gal or 5-bromo-4-chloro-3-indolyl-β-D-galactoside used in this screening method is a substrate for β-galactosidase (Figure 22.1). The enzyme β-galactosidase is the product of lacZ gene of the lac operon. It is a tetrameric protein and an initial N-terminal region (11-41) of the protein is important for activity of the protein. In this system, host contains lacZ gene without the initial region where as vector contains α-peptide to complement the defect to form active enzyme. As a result, if a vector containing α-peptide will be transformed into the host containing remaining lacz, the two fragment will reconstitute to form active enzyme. In addition, the α-peptide region in vector contains multiple cloning site and as a result of insertion of gene fragment, consequently α-peptide will not be synthesized to give fully active β-galactosidase. The enzyme β-galactosidase oxidizes x-gal to form 5-bromo-4-chloro-indoxyl and galactose. The indoxyl derivative is oxidized in air to give a blue colored dibromo-dichloro derivative (Figure 22.2). Hence, blue colored colonies indicate the presence of an active enzyme or absence of insert where as colorless colonies indicate presence of an insert.

Insertional inactivation- In this approach a foreign DNA is cloned within the coding gene responsible for a phenotype. As a result of insertion, the gene product is not available to modulate the phenotype of the host. This approach is known as insertional inactivation, and it can be used with a suitable genetic system.