How Does Insertional Inactivation Work in Genetic Engineering?
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.
What is Meant by Insertional Inactivation?
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.
Requirements in r-DNA Technology
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.
The Use of an Insertional Inactivation Process
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.
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 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.
FAQs on Insertional Inactivation Explained: Meaning, Steps & Significance
1. What is the fundamental principle of insertional inactivation in biotechnology?
Insertional inactivation is a technique used in recombinant DNA technology to identify and select host cells that have successfully incorporated a recombinant plasmid. The core principle involves inserting a foreign DNA fragment into a specific gene, known as a selectable marker, on the plasmid. This insertion disrupts the marker gene, causing it to become non-functional or 'inactivated'. By observing the loss of the marker gene's function (e.g., loss of antibiotic resistance or color production), scientists can distinguish recombinant cells from non-recombinant ones.
2. How does blue-white screening use insertional inactivation to identify recombinant bacteria?
Blue-white screening is a classic example of insertional inactivation. It uses a plasmid containing the lacZ gene, which codes for the enzyme β-galactosidase.
- When the lacZ gene is functional, the bacteria produce β-galactosidase, which breaks down a chromogenic substrate (like X-gal) in the medium, forming blue-coloured colonies.
- If a foreign gene is inserted into the lacZ gene, the gene gets inactivated.
- As a result, the recombinant bacteria cannot produce functional β-galactosidase and are unable to break down the substrate, leading to the formation of white colonies.
Therefore, white colonies represent the recombinant cells, while blue colonies are non-recombinants.
3. What is the importance of plasmids like pBR322 in demonstrating insertional inactivation?
The plasmid pBR322 is a key example for explaining insertional inactivation using antibiotic resistance. It contains two different antibiotic resistance genes: one for tetracycline (tetR) and one for ampicillin (ampR). If a foreign DNA fragment is inserted into the tetR gene, the plasmid loses its resistance to tetracycline but retains resistance to ampicillin. This allows for a two-step selection process to identify recombinants, which will grow on ampicillin plates but not on tetracycline plates.
4. What is the role of a chromogenic substrate in the context of insertional inactivation?
A chromogenic substrate is a colourless compound that produces a coloured product when acted upon by a specific enzyme. In insertional inactivation (specifically blue-white screening), its role is to provide a visual indicator of enzyme activity. For instance, X-gal is the chromogenic substrate for the enzyme β-galactosidase. When the enzyme is active (in non-recombinants), it cleaves X-gal to produce a blue pigment. When the enzyme is inactive due to gene insertion (in recombinants), no color is produced, and the colonies remain white.
5. What are the main advantages of using insertional inactivation for screening in genetic engineering?
The primary advantage of insertional inactivation is that it provides a powerful and often visual method for differentiating between recombinant and non-recombinant host cells. Instead of just confirming transformation (that the cell took up a plasmid), it specifically identifies cells that contain the plasmid with the desired foreign DNA insert. The blue-white screening method, in particular, is advantageous because it avoids the cumbersome process of replica plating required for selection with two antibiotic markers.
6. Why is blue-white screening generally considered a more efficient selection method than using two antibiotic resistance markers like in pBR322?
Blue-white screening is considered more efficient because it allows for the direct, one-step visual identification of recombinants on a single plate. In contrast, using a vector like pBR322 requires a two-step process called replica plating. First, all transformants are grown on a medium containing ampicillin. Then, these colonies must be transferred in the exact same pattern to another plate containing tetracycline. Identifying the colonies that did not grow on the second plate is cumbersome, time-consuming, and prone to error. The single-plate visual differentiation of blue and white colonies is much simpler and faster.
7. What would be the outcome if the foreign DNA was inserted into the plasmid at a location other than a selectable marker gene?
If the foreign DNA were inserted into a plasmid at a site that is not part of a selectable marker gene, the process of insertional inactivation would not occur. The selectable marker (e.g., lacZ or tetR) would remain fully functional. Consequently, the host bacterium would exhibit the traits of a non-recombinant (e.g., it would form a blue colony or show resistance to both antibiotics). This would make it impossible to distinguish these transformed cells from the non-recombinant ones using this screening method, defeating the purpose of the technique.
8. Can any gene be used as a selectable marker for insertional inactivation, or are there specific requirements?
Not just any gene can be used. A gene must have specific characteristics to be an effective selectable marker for insertional inactivation:
- It must produce a readily observable phenotype, such as antibiotic resistance or a color change, that is easy to screen for.
- The gene must contain one or more unique restriction sites within its coding sequence. This allows a foreign DNA fragment, cut with the same restriction enzyme, to be inserted specifically into that gene, ensuring its inactivation.
- The loss of its function upon insertion must not be lethal to the host cell, allowing the recombinant cells to survive for identification.
