4. Biotechnology: Principles & Processes

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Biotechnology deals with techniques of using live organisms or their enzymes for products and 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 analogues for products and services’.

Biotechnology deals with:

-    Microbe-mediated processes (making curd, bread, wine etc).

-    In vitro fertilisation (‘test-tube’ baby programme)

-    Synthesis and using of a gene

-    Preparation of a DNA vaccine

-    Correcting a defective gene

PRINCIPLES OF BIOTECHNOLOGY

The two core techniques of modern biotechnology are:

a. Genetic engineering: The technique in which the genetic material (DNA & RNA) is chemically altered and introduced into host organisms to change the phenotype.

b.Maintenance of sterile ambience: It is necessary in chemical engineering processes for growing only the desired microbe/ eukaryotic cell in large quantities for the manufacture of antibiotics, vaccines, enzymes, etc.

-    Traditional hybridisation techniques lead to inclusion and multiplication of undesirable genes along with desired genes. Genetic engineering helps to isolate and introduce only desirable genes into the target organism.

-    A piece of DNA is not able to multiply itself in the progeny cells of the organism. But, when it gets integrated into the recipient genome, it multiplies and inherits along with the host DNA.

-    First recombinant DNA was emerged from the possibility of linking a gene of antibiotic resistance with a native plasmid of Salmonella typhimurium. Stanley Cohen & Herbert Boyer (1972) isolated the antibiotic resistance gene by cutting out a piece of DNA from a plasmid.

3 basic steps in genetically modifying an organism:

a)    Identification of DNA with desirable genes

b)   Introduction of the identified DNA into the host

c)    Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

1. Restriction Enzymes (‘molecular scissors’)

-    In 1963, two enzymes responsible for restricting the growth of bacteriophage in E. coli were isolated. One of these added methyl groups to DNA. The other (restriction endonuclease) cut DNA.

-    The first restriction endonuclease is Hind II. It always cuts DNA molecules at a particular point by recognizing a specific sequence of six base pairs. This is known as the recognition sequence for Hind II.

-    Today more than 900 restriction enzymes have been isolated from over 230 strains of bacteria.

-    Naming of the restriction enzymes: First letter indicates genus and the second two letters indicate species of the prokaryotic cell from which they were isolated.

E.g. 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).

-    Restriction enzymes belong to a class of enzymes called nucleases. They include exonucleases & endonucleases.

Exonucleases

They remove nucleotides from the ends of the DNA.

Endonucleases

-    They cut at specific positions within the DNA. (For figures see TB page: 196).

-    Each restriction endonuclease can bind to specific recognition sequence of the DNA and cut each of the two strands at specific points in their sugar-phosphate backbones. Each restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA.

-    The palindrome in DNA is a sequence of base pairs that read the same on the two strands in 5' → 3' direction and in 3' → 5' direction. E.g.

                     5' —— GAATTC —— 3'

3' —— CTTAAG —— 5'

-    Restriction enzymes cut the strand a little away from the centre 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 action of the enzyme DNA ligase. (For figure see TB page: 197).

-    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.

Separation and isolation of DNA fragments:

-    DNA fragments formed by restriction endonucleases can be separated by a technique called gel electrophoresis. (For figure see TB page: 198).

-    DNA fragments are negatively charged. So they can be separated by moving them towards the anode under an electric field through a medium/matrix such as agarose (a natural polymer extracted from sea weeds).

-    The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. The smaller sized fragment move farther.

-    The separated DNA fragments can be visualized after staining the DNA with ethidium bromide followed by exposure to UV radiation. Bright orange coloured DNA bands can be seen.

-    The separated DNA bands are cut out from agarose gel and extracted from gel piece. This step is called elution. These purified DNA fragments are used in constructing recombinant DNA by joining them with cloning vectors.

2. Cloning Vectors

-    They are the DNA molecules that can carry a foreign DNA segment and replicate inside the host cells.

E.g. Plasmids (circular extra-chromosomal DNA of bacteria) and bacteriophages.

-    Bacteriophages (high number per cell) have very high copy numbers of their genome within the bacterial cells. Some plasmids have only 1-2 copies per cell. Others may have 15-100 copies per cell.

-    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 of cloning vector:

a. Origin of replication (ori): This is a sequence from where replication starts. A piece of DNA linked to ori can replicate within the host cells. This also controls the copy number of the linked DNA. So, for getting many copies of the target DNA it should be cloned in a vector whose origin support high copy number.

b.Selectable marker (marker gene): It helps to select the transformants and eliminate the non-transformants.

Transformation is a procedure in which a piece of DNA is introduced in a host bacterium.

Selectable markers of E. coli include the genes encoding resistance to antibiotics like ampicillin, chloramphenicol, tetracycline or kanamycin, etc. The normal E. coli cells do not carry resistance against any of these antibiotics.

c. Cloning sites: In order to link the alien DNA, the vector needs very few recognition sites for restriction enzymes.

Presence of more than one recognition sites generates several fragments, which complicates the gene cloning.

The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes. E.g. ligation of a foreign DNA at the Bam H I site of tetracycline resistance gene in the vector pBR322.

-    The recombinant plasmids lose tetracycline resistance due to insertion of foreign DNA. But they can be selected out from non-recombinant ones by plating the transformants on ampicillin containing medium.

-    Then these transformants are transferred on tetracycline medium. The recombinants grow in ampicillin medium but not on tetracycline medium. But, non-recombinants will grow on the medium containing both the antibiotics.

-    In this case, one antibiotic resistance gene helps to select the transformants, whereas the other antibiotic resistance gene gets inactivated due to insertion of alien DNA, and helps in selection of recombinants.

-    Selection of recombinants due to inactivation of antibiotics requires simultaneous plating on 2 plates having different antibiotics. Therefore, alternative selectable markers have developed to differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate.

-    A recombinant DNA is inserted within the coding sequence of an enzyme, â-galactosidase. So the enzyme is inactivated. It is called insertional inactivation. Such colonies do not produce any colour. These are identified as recombinant colonies.

-    If the plasmid in bacteria have no an insert it gives blue coloured colonies in presence of chromogenic substrate.

d.Vectors for cloning genes in plants and animals:

Genetic tools of some pathogens can be transformed into useful vectors for delivering genes to plants & animals. E.g.

·   Agrobacterioum tumifaciens (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. tumifaciens is modified into a cloning vector which is not pathogenic to the plants but is able to use the mechanisms to deliver genes of interest into 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 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 the 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: which when infect the cell, transfer the recombinant DNA into the host.

PROCESSES OF RECOMBINANT DNA TECHNOLOGY

1. Isolation of the Genetic Material (DNA)

-    To get pure DNA (free from other macro-molecules), the bacterial cells/plant or animal tissue are treated with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus) etc. The cell is broken to release DNA along with other macromolecules (RNA, proteins, polysaccharides and lipids).

-    Genes (DNA) are interwined with proteins such as histones. RNA is removed by treating 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

-    Restriction enzyme digestions are performed by incubating purified DNA with the restriction enzyme, at the optimal conditions.

-    Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion. As DNA is negatively charged, it moves towards the anode. The process is repeated with the vector DNA also. (For figure see TB page: 198).

-    After cutting the source DNA and the vector DNA, the cut out gene (DNA segment) of interest from the source DNA and the cut vector are mixed and ligase is added. This 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.

-    The enzyme extends the primers using the nucleotides and the genomic DNA (template). Through continuous DNA replication, the DNA segment is amplified up to 1 billion copies. (For figures see TB page: 202).

-    For amplification a thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus) is used. It remains active in high temperature during the denaturation of double stranded DNA.

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

4. Insertion of Recombinant DNA into the Host Cell/Organism

-    There are several methods of introducing the ligated DNA into recipient cells. Recipient cells take up DNA present in its surrounding.

-    If a recombinant DNA bearing ampicillin resistant gene (a selectable marker 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 ultimate aim of recombinant DNA technology is to produce a desirable protein. The foreign gene gets expressed under appropriate conditions.

-    If a protein encoding gene is expressed in a heterologous host, it is called a recombinant protein.

-    The cells with foreign genes may be grown on a small scale in the laboratory. The cultures may be used to extract the desired protein and purify it by using different separation techniques.

-    The cells can also be multiplied in a continuous culture system. Here, the used medium is drained out from one side while fresh medium is added from the other. It maintains the cells more physiologically active and so produces a larger biomass leading to higher yields of desired protein.

Bioreactors

-    To produce large quantities of products, the bioreactors are used where large volumes (100-1000 litres) of culture can be processed.

-    Bioreactors are the vessels in which raw materials are biologically converted into specific products, enzymes etc., using microbial plant, animal or human cells.

-    A bioreactor provides the optimal growth conditions (temperature, pH, substrate, salts, vitamins, oxygen) for achieving the desired product.

-    The most commonly used bioreacters are of stirring type (stirred-tank reactor) (For figures see TB page: 204).

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. 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 as in case of drugs. Strict quality control testing for each product is also required.

-    The downstream processing and quality control testing vary from product to product..

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