(1) Insertional Inactivation of antibiotic resistance gene- As discussed in an earlier lecture, bacterial plasmid PBR322 has two antibiotic resistance gene, Ap^r and Tc^r. If a gene fragment will be cloned in ScaI, it will disrupt the Ap^r gene. As a result, the clone will be ampicillin sensitive and Tc^r. where as the original plasmid will be Ap^r and Tc^r. To select the clone, first the transformed e.coli is plated on tetracycline containing media. Subsequently, a replica plate will be made on ampicillin containing media to identify the clone growing on tetracycline media but not on ampicillin media. This approach is schematically depicted in Figure 22.3.

Figure 22.3: Insertional Inactivation of antibiotic resistance gene in pBR322 to screen recombinant clone.

(2) Insertional Inactivation of LacZ gene- LacZ is a part of lac operon and responsible for synthesis of β-galactosidase. As discussed earlier, X-gal system can be used to detect the insertional inactivation of LacZ gene to screen the cloned fragment. If the gene is inserted into the lacz, the clone will not be able to produce a functional β-galactosidase. Hence, blue colored colonies indicate the presence of an active enzyme or absence of insert where as colorless colonies indicate presence of an insert. This approach is schematically depicted in Figure 22.4.