NEET Biology Class 12 Biotechnology Principles And Process Notes

Biotechnology  Principles And Process

• Biotechnology is the technique of using live organisms or their enzymes for products & processes useful to humans.
• The European Federation of Biotechnology (EFB)defines
• Biotechnology as ‘the integration of natural science and organisms, cells, parts thereof, and molecular analogs for products and services’.

Biotechnology deals with:

• Microbe-mediated processes (making curd, bread, wine etc).
• In vitro fertilization (test-tube baby program).
• Synthesis and use of a gene.
• Preparation of DNA vaccine.
• Correcting a defective gene.

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Principles Of Biotechnology

Core techniques of modern biotechnology:

• Genetic engineering: The technique in which genetic material (DNA & RNA) is chemically altered and introduced into host organisms to change the phenotype.
• Maintenance of sterile ambiance: It is necessary in chemical engineering processes for growing desired microbe/eukaryotic cell for the manufacture of antibiotics, vaccines, enzymes, etc.

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Basic steps in genetically modifying an organism:

• Identification of DNA with desirable genes: Traditional hybridization techniques lead to the inclusion and multiplication of undesirable genes along with desired genes. Genetic engineering helps to isolate and introduce only desirable genes into the target organism.
• Introduction of the identified DNA into the host: A vector DNA such as a plasmid is used to deliver an alien piece of DNA into the host organism.
• Maintenance of introduced DNA in the host and transfer of the DNA to its progeny:  A piece of alien DNA has no the sequence called Origin of replication (ori) needed for starting replication. So, it cannot multiply itself in the progeny cells of the organism. Hence alien DNA is integrated into the recipient genome (it has ori). It multiplies & inherits along with host DNA.

The first recombinant DNA (rDNA) was produced by Stanley Cohen & Herbert Boyer (1972). They isolated an antibiotic resistance gene by cutting out a DNA piece from a plasmid. This gene was linked with a native plasmid of Salmonella typhimurium.

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Tools Of Recombinant DNA Technology

1. Restriction Enzymes (‘molecular scissors’):

These are the enzymes that cut DNA at specific sites into fragments.

• They belong to a class of enzymes called nucleases. In 1963, two enzymes responsible for restricting the growth of bacteriophage in E. coli were isolated.
• One enzyme added methyl groups to DNA. The other (restriction endonuclease) cut DNA.
• More than 900 restriction enzymes have been isolated from over 230 strains of bacteria.

Naming of the restriction enzymes:

• The first letter indicates genus and the second two letters indicate species of the prokaryotic cell from which they were isolated. for example, EcoRI comes from E. coli RY 13 (R = the strain.
• Roman numbers = The order in which the enzymes were isolated from that strain of bacteria).

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Types of Restriction Enzymes:

Exonucleases: They remove nucleotides from the ends of the DNA.

Endonucleases: They cut at specific positions within the DNA.

• They bind to specific recognition sequences of the DNA and cut the two strands at specific points.
• The first restriction endonuclease is Hind II. It cuts DNA molecules by recognizing a specific sequence of 6 base pairs. This is called the recognition sequence for Hind II.
• Restriction endonuclease recognizes a specific palindromic nucleotide sequence in the DNA.
• It is a sequence of base pairs that read the same on the two strands in the 5′ → 3′ direction and in the 3′ → 5′ direction.

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For example:

5′ —— GAATTC —— 3′

3′ —— CTTAAG —— 5′

Steps in the formation of recombinant DNA by action of restriction endonuclease enzyme EcoRI

• Restriction enzymes cut the strand a little away from the center of the palindrome sites but between the same two bases on the opposite strands.
• This leaves single-stranded overhanging stretches at the ends. They are called sticky ends. They form H-bonds with their complementary cut counterparts. This stickiness facilitates the action of the enzyme DNA ligase.
• When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of sticky-ends and these are joined together by DNA ligases.

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Separation and isolation of DNA fragments

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DNA fragments are separated by a technique called gel electrophoresis.

• DNA fragments can be seen as bright orange colored bands when they are stained with ethidium bromide and exposed to UV radiation.
• DNA bands are cut out from agarose gel. This is called elution. These purified DNA are used to construct recombinant DNA by joining them with cloning vectors.

2. Cloning Vector

It is a DNA molecule that can carry a foreign DNA segment and replicate inside the host cells.

• Plasmids, bacteriophages, etc.
• Plasmids are autonomously replicating the circular extrachromosomal DNA of bacteria. Some plasmids have only
• 1-2 copies per cell. Others have 15-100 copies per cell.
• Bacteriophages (high number per cell) have very high copy numbers of their genome within the bacterial cells.
• When the cloning vectors are multiplied in the host, the
• Linked piece of DNA is also multiplied to the numbers equal to the copy number of the vectors.

Features required for cloning into a vector:

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1. Origin of replication (ori):

• This is a sequence where replication starts.
• A piece of DNA linked to or can replicate within the host cells. This also controls the copy number of linked DNA.
• So, to get many copies of the target DNA, it should be cloned in a vector whose origin support high copy number

2. Selectable marker (marker gene):

• It is a gene that helps to select the transformants and eliminate the non-transformants.
• Transformation is a procedure through which a piece of
• DNA is introduced in a host bacterium. Such a bacterium is called a transformant. If transformation does not take place, it is non-transformant.
• Selectable markers of E. coli include the genes encoding resistance to antibiotics like ampicillin, chloramphenicol, tetracycline, kanamycin etc. Normal E. coli cells have no resistance against these antibiotics.

3. Cloning sites:

To link the alien DNA, the vector needs a single or very few recognition sites for restriction enzymes.

• More than one recognition site generates several fragments. It complicates gene cloning.
• Ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic-resistant genes. ligation of foreign DNA at the Bam HI site of the tetracycline resistance gene in vector pBRr322.

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• As a result, the recombinant plasmid is formed. If ligation does not occur, it is called a non-recombinant plasmid.

• Restriction sites:  HindIII, EcoR I, BamH I, Sal I, Pvu II, Pst I, Cla I, ori
• Antibiotic resistance genes: ampR andtetR.
• Rop: codes for the proteins involved in the replication of plasmid.
• The recombinant plasmids lose tetracycline resistance due to the insertion of foreign DNA.
• When the plasmids are introduced into E. coli cells, 3 types of cells are obtained:
  1. Non-transformants: They have no plasmid. So they are not resistant to either tetracycline or ampicillin.
  2. Transformants with non-recombinant plasmid: They are resistant to both tetracycline & ampicillin.
  3. Transformants with recombinant plasmid: They are resistant only to ampicillin.
• Recombinant plasmids can be selected from nonrecombinant ones by plating transformants on an ampicillin medium. Then the transformants are transferred on a tetracycline medium.
• The recombinants grow in an ampicillin medium but not in a tetracycline medium. However, non-recombinants grow on the medium containing both antibiotics.
• Thus, one antibiotic resistance gene helps to select the transformants. The inactivated antibiotic resistance gene helps to select recombinants.
• Selection of recombinants due to the inactivation of antibiotics requires simultaneous plating on 2 plates having different antibiotics.
• Therefore, alternative selectable markers have developed to differentiate recombinants from nonrecombinants based on their ability to produce color in the presence of a chromogenic substrate.
• In this, a recombinant DNA is inserted within the coding sequence of an enzyme, β-galactosidase. So, the enzyme is inactivated. It is called insertional inactivation.
• Such colonies do not produce any color. These are identified as recombinant colonies.
• If the plasmid in bacteria has no insert, it gives blue-colored colonies in the presence of a chromogenic substrate.

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4. Vectors for cloning genes in plants & animals:

• Genetic tools of some pathogens can be transformed into useful vectors for delivering genes to plants & animals. For example,
• Agrobacterium tumefacient (a pathogen of many dicot plants) can deliver a piece of DNA (T-DNA) to transform normal plant cells into a tumor. These tumor cells produce the chemicals required by the pathogen.
• The tumor-inducing (Ti) plasmid of A. tumefacient is modified into a cloning vector that is not pathogenic to the plants but is able to use the mechanisms to deliver genes of interest to plants.
• Retroviruses in animals can transform normal cells into cancerous cells. So, they are used to deliver desirable genes into animal cells.

3. Competent Host (For Transformation with Recombinant DNA)

• DNA is a hydrophilic molecule. So, it cannot pass through cell membranes.
• To avoid this problem, bacterial cells are treated with a specific concentration of a divalent cation (e.g. calcium).
• So, DNA enters the bacterium through pores in the cell wall.
• Such cells are incubated with recombinant DNA on ice.
• Then they are placed briefly at 420C (heat shock) and put them back on ice.
• This enables the bacteria to take up recombinant DNA

Other methods to introduce alien DNA into host cells:

• Micro-injection: In this, recombinant DNA is directly injected into the nucleus of an animal cell.
• Biolistics (gene gun): In this, cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA. This method is suitable for plants.
• ‘Disarmed pathogen’ vectors: They infect the cell and transfer the recombinant DNA into the host.

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Processes Of Recombinant DNA Technology

1. Isolation of the Genetic Material (DNA):

• The bacterial cells/plant or animal tissue are treated with enzymes like lysozyme (bacteria), cellulase (plants), chitinase (fungus) etc.
• The cell is broken releasing DNA & other macromolecules (RNA, proteins, polysaccharides and lipids).
• RNA is removed by treating it with ribonuclease. Proteins are removed by treatment with protease. Other molecules are removed by appropriate treatments.
• When chilled ethanol is added, purified DNA precipitates out as a collection of fine threads in the suspension.

2. Cutting of DNA at Specific Locations:

• Purified DNA is incubated with the restriction enzyme at optimal conditions. As a result, DNA digests.
• Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion.
• DNA is negatively charged. So it moves towards the anode. The DNA fragments separate according to their size through the sieving effect of the agarose gel (a polymer extracted from seaweeds).
• The smaller fragment moves farther. The process is repeated with the vector DNA. After cutting the source DNA and vector DNA, the cut-out gene of interest from the source.
• DNA and cut vector are mixed and ligase is added. It creates recombinant DNA.

3. Amplification of Gene of Interest using PCR:

• Polymerase Chain Reaction (PCR) is the synthesis of multiple copies of the gene of interest in vitro using 2 sets of primers & the enzyme DNA polymerase.
• Primers are small chemically synthesized oligonucleotides that are complementary to the regions of DNA.

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Steps of PCR:

• Denaturation: It is the heating of target DNA (gene of interest) at a high temperature (940 C) to separate the strands.
• Each strand acts as a template for DNA synthesis.
• Annealing: It is the joining of the two primers (at 520 C) at the 3’ end of the DNA templates.
• Extension: It is the addition of nucleotides to the primer with the help of a thermostable DNA polymerase called
• Taq polymerase:
  • It is isolated from a bacterium, Thermus aquaticus. It remains active in high temperatures during the denaturation of double-stranded
  • DNA. Through continuous replication, the DNA segment is amplified up to 1 billion copies.

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The amplified fragment can be used to ligate with a vector for further cloning.

4. Insertion of Recombinant DNA into Host Cell:

• Using any method, the ligated DNA is introduced into recipient cells. They take up DNA from its surroundings.
• If a recombinant DNA-bearing ampicillin resistant gene is transferred into E. coli cells, the host cells become ampicillin-resistant cells.
• If the transformed cells are spread on agar plates containing ampicillin, only transformants will grow.
• Untransformed recipient cells will die.

5. Obtaining the Foreign Gene Product:

• The aim of recombinant DNA technology is to produce a desirable protein.
• If a protein-encoding a foreign gene is expressed in a heterologous host, it is called a recombinant protein.
• The cells with foreign genes can be grown in the laboratory.
• The cultures are used to extract the desired protein and purify it by using separation techniques. The cells can also be multiplied in a continuous culture system.
• Here, the used medium is drained out from one side while the fresh medium is added from the other.
• It maintains the cells more physiologically active and so produces a larger biomass. It yields more desired protein.

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Bioreactors:

• These are the vessels in which raw materials are biologically converted to specific products, enzymes, etc., using microbial plant, animal, or human cells.
• Bioreactors are used to produce large quantities of products. They can process 100-1000 liters of culture.
• A bioreactor provides the optimal growth conditions (pH, temperature, substrate, salts, vitamins, oxygen) for achieving the desired product.
• The most commonly used bioreactors are of stirring type (stirred-tank reactor).

It is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability.  Alternatively, air can be bubbled through the reactor.

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The bioreactor has:

• An agitator system
• An oxygen delivery system
• A foam control system
• A temperature control system
• pH control system
• Sampling ports (for periodic withdrawal of the culture).

6. Downstream Processing:

• It is a series of processes such as separation and purification of products after the biosynthetic stage.
• The product is formulated with suitable preservatives.
• Such formulation undergoes thorough clinical trials and strict quality control testing.