Introduction to Insertional Inactivation and its Relation with Biotechnology
The introduction of biotechnology has opened the ways to many discoveries which further led to the development of many technologies that might have appeared impossible in earlier times. Genetic engineering came into existence under which the genes of an organism are altered in order to get a certain desired result in its phenotype. Also, the production of substances like antibiotics from the bacteria itself can be done today. Biotechnology made all these works possible. Insertional inactivation is one such process that comes under this stream of science. The word itself is reflecting its meaning which is inactivation caused due to insertion.
Recombinant DNA(rDNA) Technology
Before understanding the process of Insertional inactivation one must know about the rDNA technology. This is a technique of genetic engineering under which recombinant DNA is created.
Every living organism has its own genetic material DNA/RNA in each cell of its body. This genetic material has all the information on the characters that would be present in the organism.
For example in the case of humans, the information about the hair color will be present in the DNA, whether it is black/brown/golden. These kinds of genetic information are present in the small units of DNA called genes.
Altering (adding new or deleting the existing one) these genes in the DNA of an organism is known as Recombinant DNA technology and the new DNA obtained by this technique is the Recombinant DNA (rDNA).
Requirements in r-DNA Technology
There are certain tools required for carrying out the process of alteration of DNA of a particular organism. Some of them are described below.
Restriction Enzymes- These enzymes have the power to either cut a certain DNA or add certain chemical groups to a particular DNA. The one which cuts the DNA is called a Restriction endonuclease enzyme. These restriction enzymes do not cut DNA randomly but have certain recognition points. If they encounter such points then they cut the DNA at those points.
Example - EcoRI is a restriction endonuclease enzyme that cuts the DNA at site GAATTC base pairs in the DNA.
Ligase Enzymes- These are those enzymes that help in joining a particular foreign DNA segment to that DNA in which changes are required or are performed.
Vectors- These are those organisms that help in the transfer of the recombinant DNA in the host organism. Cloning vectors increase the number of recombinant DNA by multiplying.
For example bacteriophages.
Selectable Markers- These are those substances that help in the detection of the recombinant organisms and the non-recombinant organisms. Antibiotics such as tetracycline, ampicillin, etc.
This process refers to the insertion of a certain gene in the host DNA at a particular site. Due to this insertion, the function of the gene at whose site insertion has taken place gets inactivated.
Use of Insertional Inactivation Process
This process is used to identify recombinant DNA containing organisms from those who are non-recombinants. This process actually makes the appropriate use of selectable markers.
Insertional Inactivation Method
The plasmid is the main component for carrying out the process of Insertional Inactivation. A plasmid has various genes present in it on different sites. These genes offer features like antibiotic resistance to the organism that incorporates them. A plasmid named pBR322 is considered for carrying out the process or method of the Insertional Inactivation.
[Image will be Uploaded Soon]
This plasmid has two sites that give resistance towards antibiotic ampicillin and tetracycline respectively. By the technique of genetic engineering, a foreign gene is inserted in the site BamHI (site for tetracycline resistance). Now the recombinant plasmid will lose the resistance towards tetracycline as some other gene is inserted at its place. To recognize the recombinant the plating of ampicillin and tetracycline is used. Recombinant bacteria will grow in the ampicillin but will start dying in the tetracycline as they have lost tetracycline resistance.
1.What is Meant by the Insertional Inactivation of the Lacz Gene?
Insertional Inactivation of an antibiotic resistance gene and then identifying the recombinants by setting the plates of both antibiotics tetracycline and ampicillin is a very complex process.
Thus alternate selectable markers are used on the basis of color-producing ability in chromogenic substance.
In plasmid pUC19 a gene lacz is present. It encodes for the production of the beta-galactosidase enzyme.
This enzyme leads to the development of a blue color colony in the chromogenic substance when it is active.
A foreign gene is inserted in the site of this lacz gene thus deactivating it.
Now the recombinant organism will not produce beta-galactosidase enzyme as a result and no blue color colony is seen. and white colonies are developed.
Thus due to this color difference transformant and non-transformants can be identified.
2. What are Plasmids and How They are Useful in the Insertional Inactivation Process?
Plasmids are small circular DNA which is not a part of cellular DNA found in the nucleus. They are found in certain bacteria. They provide unique features to the bacteria in which they are present. They also have the ability to replicate the genomic DNA independently. Their replication number depends on the origin of replication (ori). These plasmids help in the selection of recombinants and also in the formation of recombinants. Thus due to all these properties, they are useful in the process of insertional inactivation.