Class 12 Biology Chapter 11 Biotechnology Principal and Processes Notes

We provide comprehensive notes and solutions for Class 12 Biology Chapter 11: Biotechnology Principles and Processes. Explore the fundamental concepts and practical applications of biotechnology in this chapter to enhance your understanding and excel in your examinations.

Class 12 Biology Chapter 11 Biotechnology Principal and Processes Notes

Biotechnology Principles and Processes: Biotechnology is the field of biology which is used to develop various technologies that help in the production of certain products that result in the welfare of human beings. It consists of various applications in different fields that include therapeutics, processed food, diagnostics, waste management, genetically modified crops, energy production, etc. The definition of biotechnology given by the European Federation of Biotechnology states that “The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.”

Principles of Biotechnology:

Modern biotechnology is based on two core techniques that are:

Genetic Engineering: Genetic engineering is the direct manipulation of an organism’s gene by the use of biotechnology which is used to change the genetic makeup of the cell. The set of technologies are used for the genetic makeup of the cells which includes the transfer of genes in the species boundaries for the production of improved organisms, most importantly called clones resulting in gene cloning.

Maintenance of a Sterile Environment in Chemical Engineering Processes: It helps in the growth of only those microbes that are required and this process helps in the manufacturing of vaccines, antibiotics, drugs, etc.

Basic Principles of Biotechnology:

Genetic engineering involves the isolation and introduction of only those genes into an organism that is desired and does not introduce undesirable genes. The steps involved in genetic engineering are: 

• Development of recombinant DNA (rDNA). 
• Cloning of the desired gene. 
• Transfer of the cloned gene into the suitable host organism.

Origin of Replication (ori): The sequence of chromosomes in the DNA that helps in the initiation of the relocation of DNA. The foreign DNA that is inserted into the host organism needs to be attached to the origin of relocation and this results in the formation of multiple copies of the DNA while if the foreign gene is not attached to the origin of replication then it may not result in the multiplication of DNA.

Cloning: The process of formation of several identical copies of the DNA template.

Plasmid: An extrachromosomal, circular DNA material that helps in the replication of DNA. they are used as cloning vectors and also helps in the process of gene expression. Here, a foreign gene is inserted into the plasmid which then multiplies and results in the formation of several copies of the desired gene.

Antibiotic Resistance Gene: In the case of certain microorganisms there are several genes that have the ability to grow when there is a specific antibiotic present while the genes provide resistance against them. These genes are found to be located on the plasmids and are used in the process of cloning and transformation.

Restriction Enzymes: These enzymes are responsible for the cutting of DNA fragments at specific sites, thus they are called the “molecular scissors”. These enzymes cut the DNA at a particular site that is specific for each restriction enzyme. They help in the process of cutting the sedated gene which is then inserted into the specific locations of the vector or the host DNA.

Vectors: They are the plasmids that help in the process of multiplication and then the transfer of genes from one organism to the other.

Ligase: They are those enzymes that joined together the fragrant of DNA that contains the desired gene and the DNA of the host. They help in the sticking of fragments of DNA together.

The basic steps in the genetic modification of an organism:

• Identification of desired DNA fragments.
• Introduction of desired DNA fragments into a suitable host.
• Maintaining foreign DNA in the host and its transfer to the progeny.

Tools for Genetic Engineering (Recombinant DNA Technology):

Restriction enzymes also called molecular scissors are used to simply cut the DNA which is then inserted into the vector. These restriction enzymes help in the addition of the methyl groups to the DNA that results in the restriction of the digestion of their own DNA. These enzymes cut DNA fragments at their particular recognition sequences.

Recognition Sequences: The bases of the DNA sequence that are specific for each restriction enzyme and act as the site for restriction or cutting resulting in the formation of the palindromic sequences.

There are two types of restriction enzymes: endonucleases and exonucleases.

Endonucleases: These enzymes are responsible for the cutting of the DNA in the middle while the exonucleases enzymes are responsible for the cutting of the DNA at the ends. Examples of restriction endonucleases are ECoR1, Hind III, etc. Restriction enzymes cut the DNA molecule at a specific site that is known as a restriction site. Each endonuclease characterized the restriction site by a specific recognition sequence. Each restriction endonuclease is responsible for the identification of the specific palindromic nucleotide sequence in the DNA. The Palindromic DNA sequence of the base pairs is present on the two strands of DNA in the same order when the orientation of reading is kept the same.

Ligases: Ligases are the enzyme that is responsible for the joining of the two DNA fragments. The process of ligation occurs in the presence of sticky ends (they are the similar overhanging sequences formed due to the action of the same restriction enzyme).

Palindromic nucleotide sequences: Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Once it finds its specific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones Each restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA.

Restriction Enzymes: the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated. One of these added methyl groups to DNA, while the other cut DNA. The later was called restriction endonuclease.

Separation and Isolation of DNA Fragments:

The technique called gel electrophoresis is responsible for the separation of the DNA fragments obtained through restriction.

Gel Electrophoresis: The process of migration of negatively charged DNA towards the positively charged electrode through a porous polymer gel matrix when the electric current is passed in an electric field. The DNA fragments will then start to move in the gel and will separate or resolve based on their size as well as the pore size of the gel. The smaller DNA fragments will be able to cover the larger distance while the larger DNA fragments will cover a smaller distance. They commonly use gel matrix for the process of DNA electrophoresis is agarose which is obtained from seaweeds.

Visualization: To observe the DNA fragments they first need to be stained by the compound called ethidium bromide (EtBr) since they cannot be observed directly and are then exposed to the UV light this will result in the fluoresces of DNA. 

Elution: The process of elution involves the purification of the desired DNA fragments using various methods from the gel.

Cloning Vectors:

Vector is any DNA molecule that is responsible for the carrying of the desired gene that needs to be inserted into the host organism. For example, plasmid. The plasmid is an extrachromosomal autonomously replicating genetic content that is present in the bacteria and is different from the other chromosomal DNA. It helps in the transfer of desired genes into the host cell. Plasmids consist of an origin of replication, it is the site responsible for the replication as soon as the gene of interest enters the host cell. It also contains the antibiotic resistance gene.

Following features are required for a cloning vector:

Origin of Replication: This is known as ori. This helps in the replication of DNA fragments into the host cell and results in the maintenance of the number of copies of DNA. 

Selectable Marker to Identify Transformed Cells: The process of introduction of a piece of DNA into the host cells is known as the transformation. The genes that encode resistance towards certain antibiotics such as ampicillin, chloramphenicol, tetracycline, or kanamycin, etc. are some of the useful selectable markers for E. coli and in the absence of these selectable markers, the normal E. coli cells do not show any resistance against any of these antibiotics. 

There Should be a Cloning Site in the Cloning Vector: There must be one cloning site present so as not to complicate the process of cloning. The antibiotic resistance gene present as the restriction sites are responsible for the ligation of the foreign DNA. When the desired gene is introduced at the site of the antibiotic resistance gene resulting in the loss of antibiotic resistance. This results in the loss of antibiotic resistance in the recombinant plasmid. So, recombinants can be selected from the non-recombinants. Another method is insertional inactivation which is used to find out the transformed cells. This is based on the ability to produce colour when the chromogenic substrate is present. In this technique, the recombinant DNA is introduced into the coding sequence of an enzyme, β-galactosidase. Beta-galactosidase converts galactose into lactose. If a gene is introduced into this region, the formation of the β-galactosidase will not, and thus there will be no formation of lactose resulting in the inactivation of the enzyme which is called insertional inactivation. The blue colour of the non-transformed colonies occurs due to the presence of a chromogenic substrate while no colour is produced in the colonies if the insertional inactivation of the galactosidase occurs due to the presence of the gene of interest. These colonies can be named recombinant colonies.

Insertional Inactivation: The process of introduction of the desired gene in the coding region of DNA that results in the inactivation of an enzyme.

Vectors for Cloning in Plants:

A pathogen of various dicot plants, Agrobacterium tumefaciens is used as a vector for the plants. It is responsible for carrying the piece of DNA known as ‘T-DNA’ that results in the transformation of the normal plant cells into a tumor which then results in the production of the chemicals that are required by the pathogen. The desired gene is introduced along with the other required genes into the T-DNA that result in the transformation of the plant cells. The tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens is modified into a cloning vector which is no more pathogenic to the plants. In plasmids, the growth regulator is the coding genes of the cytokinin and auxin. The sources of energy are the gene codes responsible for the catabolism of opine. The transfer of T-DNA into the required host plant cell requires the right and left borders. Similarly, in the case of animal cells, the retroviruses have been modified to act as vectors.

Competent Host:

The bacterial cells need to be competent in order to take up the DNA which can be achieved by treating the cells with a specific concentration of divalent ions such as calcium ions, which results in the formation of pores in the cell wall of the bacteria. These bacteria are prone to heat shock. In this method, the calcium-treated competent cells are kept on ice, then they are incubated briefly at 42◦C for 1-2 minutes, and then immediately placed in ice. This converts the rDNA into the competent cell. Other methods used for the insertion of DNA into the host cells are microinjection, biolistic, gene gun, etc. By the method of microinjection, the DNA can be inserted directly into the nucleus of the host cell while in the case of biolistic, a high-velocity microparticle of gold or tungsten coated with DNA is required.

Process of Recombinant DNA Technology:

There are several steps involved in the process of recombinant DNA technology. 

Isolation of the Genetic Material: The membrane surrounding the DNA needs to be removed to isolate the DNA. This can be done with the help of lysozyme enzymes that result in the breaking of the cell walls of the cells of bacteria, breaks cellulase (in case of plant cells), and chitinase (in case of fungus). The RNA can be isolated with the help of ribonucleases while proteins can be removed using proteases. Lastly, the DNA obtained is treated with ethanol so as to remove the remaining impurities. DNA is then obtained as fine threads in suspension.

Restriction Digestion of the Isolated DNA: The restriction digestion of the DNA is progressed with the help of the agarose gel electrophoresis. The desired gene is then introduced into the specific vector and is joined with the help of an enzyme known as a ligase which results in the formation of the recombinant DNA molecule.

Amplification of Gene of Interest Using PCR: The amplification of the desired gene of the DNA can be done by the process of the Polymerase chain reaction (PCR). There are two sets of primers required that are the forward primer and the reverse primer. The DNA amplification is done with the help of the DNA polymerase enzyme. Taq polymerase is the most commonly used polymerase during PCR.

Insertion of Recombinant DNA Into Host Cell or Organism: The host cells need to be more competitive so as to receive the recombinant DNA. 

Expression of Desired Protein: The main aim of the recombinant DNA technology is to obtain desired protein of interest. Thus, the protein which is obtained is known as a recombinant protein.

Bioreactors: Bioreactors are the large vessels that are used to produce large quantities of recombinant protein. To achieve the desired product the optimal growth conditions (temperature, pH, substrate, salts, vitamins, oxygen) are provided by the bioreactors.

Basic Parts of a Bioreactor:

• Agitator 
• Oxygen Control system 
• Foam control system 
• Temperature control 
• pH control 
• Sampling port 
• Inlet 
• Outlet

There are mainly two types of bioreactors: Stirred type and the sparger type.

Stirring Type Bioreactor:

The stirrer type of bioreactor consists of a stirrer that are having a curved base and functions in the better mixing of the contents. It also improves the aeration of the medium. 

Sparger Type Bioreactor: 

In the sparger type of bioreactor, the air is bubbled that is generated from the base of the bioreactor which results in the mixing as well as aeration of the contents.

Downstream Processing:

The downstream processing involves those processes and methods that are responsible for the separation and purification of the desired product. The products produced in the case of drugs need to be formulated suitably and also the drugs need to be tested before they are made available commercially.