Inactivation is a recombinant DNA technology technique used to select bacteria that contain recombinant plasmids. In gene cloning, it is frequently used to identify recombinant vectors and, as a result, to distinguish a recombinant carrier from a non-recombinant vector. Inserting a fragment of foreign DNA into a cloning site on antibiotic-resistant genes on the vector, for example, can result in insertional inactivation, resulting in the loss of the antibiotic resistance phenotype. The recombinant vector will specify antibiotic sensitivity, while the non-recombinant vector will specify antibiotic resistance.
Inactivation rate
The ultimate rate of enzyme inactivation is defined by the first-order rate constant. This is called the Inactivation rate. The constant unit is written as min-1
Requirements for rDNA Technology
To change an organism’s DNA, specific pre-requisites are needed. Here are a few instances.
Restriction Enzymes: A variety of enzymes can cut a specific DNA strand or add compounds to a specific DNA strand. Cutting DNA is done by a restriction endonuclease enzyme. Restrictions Endonucleases attack DNA at specific locations rather than cutting it at random. Instead, they break DNA at them when they come upon certain spots. EcoRI, a restriction endonuclease enzyme, cuts DNA at position GAATTC is an excellent example of Restriction enzymes.
Ligase Enzymes: This type of enzyme aids in the joining of a foreign DNA fragment to DNA where alterations are needed or are being conducted.
Vectors: These organisms transfer recombinant DNA into the host organism. Cloning vectors produce a higher volume of recombinant DNA by replicating. Bacteriophages are an excellent illustration of this.
Selectable Markers- Substances like this help distinguish between recombinant and non-recombinant organisms. There are other antibiotics on the market, including tetracycline and ampicillin.
Insertional Inactivation method
- The plasmid is the most critical component in the Insertional Inactivation process. Various genes are present at different locations in a plasmid. Antibiotic resistance is one of the features that these genes provide to the species that adopt them. The plasmid pBR322 is being considered in the Insertional Inactivation process or method.
- This plasmid has two elements that confer antibiotic resistance to ampicillin and tetracycline. A foreign gene is put into the BamHI site using the genetic engineering approach (site for tetracycline resistance). Because a different gene has been added in its place, the recombinant plasmid can no longer be resistant to tetracycline. Ampicillin and tetracycline plating are used to identify the recombinant. Because recombinant bacteria have lost their tetracycline resistance, they will grow in ampicillin but die in tetracycline.
- The lacZ gene, which is a reporting gene, is inserted into the vector as an insert. The lacZ gene encodes an a-galactosidase enzyme with a few restriction enzyme recognition sites. The synthesised substrate X-gal, also known as BCIG (5-Bromo-4-chloro-indolyl—D-galactopyranoside), is transformed into an insoluble blue product as a result of this reaction.
- When a foreign gene is introduced into lacZ, the gene is silenced. No blue colour will develop as a result of lacZ deactivation since no -galactosidase will be generated. On an X-gal media, a host cell harbouring rDNA tends to create white colonies, whereas a host cell containing non-recombinant DNA creates blue colonies. As a result, the colony’s colour is used to select the recombinants.
Conclusion
The procedure of inactivating a resistance gene & subsequently detecting recombinants by using tetracycline and ampicillin plates is complicated. As a result, alternately selected markers are utilised based on the ability of chromogenic chemicals to produce colour. Insertion of rDNA into the coding sequence of alpha-galactosidase inactivates the enzymes, a process known as insertional inactivation. Recombinant microorganisms do not develop blue colonies in the vicinity of a chromogenic medium, but non-recombinant bacteria do.