A recombinant DNA technique called inactivation involves the insertional inactivation of DNA. By using this method, recombinant plasmids or fragments of foreign DNA are added to bacteria to insert into a restriction site inside a gene to be resistant to antibiotics. This results in the gene becoming inactive or non-functioning as a result.
Upon the insertion of a foreign gene into a pUC19 plasmid vector, the lacZ gene which encodes beta-galactosidase can no longer be produced. A fundamental process of screening and selection of recombinant DNA occurs once the molecule is inserted into the host cell when it becomes necessary to identify those cells containing the molecule.
Selection or screening are terms used to describe this process. In virtue of this, certain traits or characteristics are either not expressed or not expressed. The process of inactivating elements is an effective method of screening. During this procedure, foreign DNA is introduced to disturb one of the genetic characteristics. It is often referred to as the Blue-White selection method because it is widely used in the selection of recombinant plasmids for inactivation procedures.
An insert is made in the vector containing the lacZ gene, which is a reporter gene. There are a few restriction enzyme recognition sites in the β-galactosidase enzyme encoded by the lacZ gene. As a result of this reaction, a synthetic substrate X-gal, also called BCIG (5-Bromo-4-chloro-indolyl-β-D-galactopyranoside) is converted into an insoluble product with a blue color.
In the case of introducing a foreign gene to lacZ, the gene will be deactivated. Due to the deactivation of lacZ, no blue color will develop as no β-galactosidase will be produced. Thus, a host cell that contains rDNA tends to produce white-colored colonies on an X-gal medium, while a host cell carrying non-recombinant DNA tends to produce blue-colored colonies. Therefore, the color of the colony is used to select the recombinants.
In order to alter the DNA of an organism, certain tools must be used. Following are some examples.
Restriction Enzymes- A number of enzymes have the ability to either cut a particular DNA strand or to add chemical groups to a specific DNA. A restriction endonuclease enzyme is responsible for cutting DNA. Restrictions Endonucleases attack DNA at specific points and do not randomly cut it. Instead, they cut DNA at these points when they come across such points.
Example- Using EcoRI, DNA is cut at site GAATTC, where it is a restriction endonuclease enzyme.
Ligase Enzymes- An enzyme such as this helps join a foreign DNA segment to DNA where changes need to be made or are being performed.
Vectors- Recombinant DNA is transferred into the host organism by these organisms. By multiplying, cloning vectors produce a greater amount of recombinant DNA.
Example: Bacteriophages are a good example of this.
Selectable Markers- Substances such as these aid in detecting recombinant organisms and non-recombinant organisms. A number of antibiotics such as tetracycline and ampicillin are available on the market.
In this process, recombinant DNA-containing organisms are distinguished from non-recombinant ones. Markers are selected based on their ability to determine recombinant DNA content.
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 Insertional Inactivation.
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This plasmid has two sites that give resistance to the antibiotics 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 are the advantages of insertional inactivation?
During this process, the gene at which the insertion has occurred loses the function it had before. A recombinant DNA-containing organism is identified from those that do not contain recombinant DNA by using this approach. In reality, selectable markers are used to identify these organisms.
2. How is beta-galactosidase inactivated by insertional inactivation?
In the field of recombinant DNA technology, inactivation by insertion is a common process. Nevertheless, the lacZ gene gets inactivated when it is inserted into a foreign gene, such as a recombinant plasmid. The deactivation of the lacZ gene results in no blue color colonies developing.
3. What is insertional inactivation used for?
In the case of alpha-galactosidase, insertion of rDNA into its coding sequence inactivates the enzyme, a process called insertional inactivation. In the presence of a chromogenic substrate, recombinant bacteria do not produce blue colonies, while non-recombinant bacteria produce blue colonies.
4. What is the purpose of insertional inactivation as a selectable marker to differentiate recombinant from non-0recombinant proteins?
Alpha-galactosidase is inactivated by insertional inactivation, which is caused by the insertion of rDNA into its coding sequence. In the presence of a chromogenic substrate, the recombinant bacteria do not produce a blue color while the non-recombinant bacteria produce a blue color.
5. What are Plasmids and how are they used in insertional inactivation?
Platy Nuclei are small circular DNA molecules that are not a part of the nucleus. Various types of bacteria possess them. Their presence confers special characteristics on the bacteria that harbor them. Moreover, they are capable of independently replicating the genome DNA. The number of copies of genome DNA depends on the origin of replication (ori). In addition to enabling recombinant selection, these plasmids also enable recombinant formation. They are therefore useful in the process of insertional inactivation because they possess all these properties.
6. What are Plasmids and how they are useful in the Insertional Inactivation Process?
Plasmids are small circular DNA that 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.
7. 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 substances.
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 transformants and non-transformants can be identified.