Insertional inactivation
In principle, when a foreign gene (or DNA fragment) is inserted into a site within a gene, the gene loses its original function, which is called insertion inactivation. Insertion inactivation method is different from Recombinant DNA The main method to identify the transformants containing the target gene (screening) from the transformants obtained by molecules. The foreign DNA fragment is inserted into the Polyclonal site After that, the marker gene will be inactivated, showing that the corresponding antibiotic resistance of the transformant disappears or the color of the transformant changes, through which it can be preliminarily identified that the transformant is a recombinant or a non recombinant. Commonly used β - galactosidase Color development method Blue white screening method 。 1. Insertion inactivation screening of resistance genes
according to Antibiotic resistance gene The insertion inactivation method designed according to the insertion inactivation principle is a common screening method for recombinant. For example, tetracycline and Ampicillin The resistance genes are normal, and the phenotype is AprTcr. The receptor bacteria with this plasmid can grow on the double resistant plate added with tetracycline and ampicillin. However, if foreign fragments are inserted into the tetracycline resistance gene of the plasmid, the tetracycline resistance gene will be inactivated and become AprTcs. The host bacteria carrying this plasmid can grow on the ampicillin plate, but not on the tetracycline resistance plate. according to Antibiotic resistance gene The insertion inactivation method designed based on the insertion inactivation principle requires photocopying of the colony plate to select the required recombinant, which greatly increases the screening workload. Later, people designed the production of β - galactosidase as the carrier of color screening markers, which simplified the screening procedure and improved the sensitivity. Such carrier systems include M13 bacteriophage PUC plasmid system, pEGM plasmid system. Their common feature is that the carrier carries a section of lacZ gene of bacteria, which encodes a 146 amino acid alpha peptide of β - galactosidase. The receptor bacteria transformed by the carrier is lacZ Δ M15 genotype. In this way, the carrier has complete β - galactosidase activity through complementation with the host. If exogenous DNA is inserted into the lacZ gene of the vector, the lacZ gene will be inactivated, unable to synthesize α - peptide, lose the complementation of the host, unable to form functional β - galactosidase, and lose the ability to decompose X-gal. On the X-gal plate, the bacteria containing the positive recombinant are chromogenic colonies (plasmid vectors) or colorless plaque (M13 Bacteriophage carrier ); The bacteria transformed by non recombinant bacteria are blue colonies or blue plaque. The screening method of color reaction is relatively simple, but the synthesis of β - galactosidase needs induction. In the experiment, the comforting inducer IPTG (isopropyl thio - β - D-galactoside) plays an inducing role, and the acting substrate is X-gal (5-bromo-4-chloro-3-indole - β - D-galactoside). Generally, X-gal and IPTG are mixed and coated on the surface of solid plate. 
