Superb Learning Destination 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 arecombinant 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..5. Biotechnology and its applications
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Biotechnology has a wide range application such as biopharmaceuticals, therapeutics, diagnostics, genetically modified crops for agriculture, processed food, bioremediation, waste treatment and energy production.
a. Providing the best catalyst in the form of improved organism usually a microbe or pure enzyme.
b. Creating optimal conditions through engineering for a catalyst to act.
c. Downstream processing technologies to purify the protein/organic compound.
APPLICATIONS IN AGRICULTURE
Three options for increasing food production
a. Agro-chemical based agriculture
b. Organic agriculture
c. Genetically engineered crop-based agriculture
Genetically Modified Organisms (GMO) or transgenic organisms are the plants, bacteria, fungi & animals whose genes are altered by manipulation.
Advantages of genetic modification in plants:
· It makes crops more tolerant to abiotic stresses (cold, drought, salt, heat etc).
· Pest-resistant crops reduce the use of chemical pesticides.
· It helps to reduce post harvest losses.
· It increases efficiency of mineral usage by plants (this prevents early exhaustion of fertility of soil).
· It enhances nutritional value of food. E.g. Vitamin ‘A’ enriched rice.
· GM is used to create tailor-made plants to supply alternative resources to industries, in the form of starches, fuels and pharmaceuticals.
Pest Resistant Plants
- Pest Resistant Plants act as bio-pesticide.
- It reduces the need for insecticides.
- E.g. Bt cotton, Bt corn, rice, tomato, potato, soyabean etc.
Bt Cotton:
- Some strains of Bacillus thuringiensis have proteins that kill insects like coleopterans (beetles) lepidopterans (tobacco budworm, armyworm) & dipterans (flies, mosquitoes).
- B. thuringiensis forms a toxic insecticidal protein (Bt toxin) crystal during a particular phase of their growth. It does not kill the Bacillus as it exists as inactive protoxins.
- When an insect ingest the inactive toxin, it is converted into active toxin due to the alkaline pH of the gut which solubilise the crystals. The toxin binds to the surface of midgut epithelial cells and creates pores. It causes cell swelling and lysis and death of the insect.
- Bt toxin genes were isolated from B. thuringiensis and incorporated into crop plants such as cotton.
- Most Bt toxins are insect-group specific. The toxin is coded by a gene named cry. E.g. the proteins encoded by the genes cryIAc and cryIIAb control the cotton bollworms that of cryIAb controls corn borer.
Nematode resistance in tobacco plants:
- A nematode Meloidegyne incognitia infects the roots of tobacco plants and causes a great reduction in yield.
- RNA interference (RNAi) strategy is used to prevent this infestation.
- RNAi is a method of cellular defense in all eukaryotic organisms. It prevents translation of a specific mRNA (silencing) due to a complementary dsRNA molecule.
- The source of this complementary RNA is from an infection by RNA viruses or mobile genetic elements (transposons) that replicate via an RNA intermediate.
- Using Agrobacterium vectors, nematode-specific genes (DNA) were introduced into the host plant. It produced both sense & anti-sense RNA in host cells. These two RNA’s being complementary to each other formed a double stranded (dsRNA) that initiated RNAi and thus, silenced the specific mRNA of nematode. Thus the parasite cannot survive in a transgenic host expressing specific interfering RNA.
APPLICATIONS IN MEDICINE
- The recombinant DNA technology helps for the mass production of safe and more effective therapeutic drugs.
- The recombinant therapeutics does not induce unwanted immunological responses as is common in case of similar products isolated from non-human sources.
- At present, about 30 recombinant therapeutics have been approved for human-use. In India, 12 of these are presently being marketed.
Genetically Engineered Insulin:
- Management of adult-onset diabetes is possible by taking insulin at regular time intervals.
- Now, it is possible to produce human insulin using bacteria.
- Insulin from the pancreas of animals (cattle & pigs) causes allergy or other types of reactions to the foreign protein.
- Insulin consists of two short polypeptide chains (chain A & chain B) that are linked together by disulphide bridges.
- In mammals, insulin is synthesized as a pro-hormone. The pro-hormone needs processing before it becomes a fully mature and functional hormone.
- The pro-hormone contains an extra stretch called the C peptide. This is removed during maturation into insulin.
- In 1983, Eli Lilly an American company prepared two DNA sequences corresponding to A & B chains of human insulin and introduced them in plasmids of E. coli to produce insulin chains. Chains A & B were produced separately, extracted and combined by creating disulfide bonds to form human insulin.
Gene Therapy:
- It is a method to correct a gene defect diagnosed in a child/embryo. Here, genes are inserted into a person’s cells and tissues to treat a hereditary disease. It compensates for the non-functional gene.
- First clinical gene therapy was given in 1990 to a 4-year old girl with adenosine deaminase (ADA) deficiency. The disorder is caused due to the deletion of the gene for adenosine deaminase (the enzyme crucial for the immune system to function).
- This can be cured by bone marrow transplantation or by enzyme replacement therapy (injection of functional ADA). But these approaches are not completely curative.
- In gene therapy, lymphocytes from the patient’s blood are grown in a culture. Then, a functional ADA cDNA (using a retroviral vector) is introduced into these lymphocytes. Then, they are returned to the patient. This should be periodically repeated as these cells are not immortal. However, if the ADA gene (from marrow cells) is introduced into cells at early embryonic stages, it could be a permanent cure.
Molecular Diagnosis
- Recombinant DNA technology, PCR and Enzyme Linked Immuno-sorbent Assay (ELISA) are some techniques for early diagnosis.
- Presence of a pathogen is normally suspected only when the pathogen has produced a symptom. By this time the concentration of pathogen is already very high in the body. However, very low concentration of a bacteria or virus can be detected by amplification of their nucleic acid by PCR.
- PCR is used to detect HIV in suspected AIDS patients. It is also used to detect mutations in genes in suspected cancer patients. It is a powerful technique to identify many other genetic disorders.
- A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is allowed to hybridise to its complementary DNA in a clone of cells followed by detection using autoradiography. The clone having the mutated gene will hence not appear on the photographic film, because the probe will not have complimentarity with the mutated gene.
- ELISA is based on the principle of antigen-antibody interaction. Infection by pathogen can be detected by the presence of antigens (proteins, glycoproteins, etc.) or by detecting the antibodies synthesized against the pathogen.
TRANSGENIC ANIMALS
- These are the animals whose genome has been altered by introduction of an extra (foreign) gene by manipulation.
- E.g. Transgenic rats, rabbits, pigs, sheep, cows and fish.
- Over 95% of all existing transgenic animals are mice.
Benefits of transgenic animals
· To study normal physiology & development: Transgenic animals are used to study how genes are regulated, and how they affect the normal body functions and its development.
E.g. study of complex factors such as insulin-like growth factor. Genes (from other species) that alter the formation of this factor are introduced and the biological effects are studied. This gives information about the biological role of the factor in the body.
· To Study the contribution of genes in the development of a disease: Transgenic models help for investigation of new treatments for human diseases. E.g. transgenic models for many human diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer’s.
· Biological products: Some medicines contain biological products, but they are often expensive. Transgenic animals are used to produce useful biological products by introducing genes which codes for a particular product.
E.g. human protein (a-1-antitrypsin) used to treat emphysema, products for treatment of phenylketonuria (PKU) and cystic fibrosis etc.
In 1997, Rosie (first transgenic cow) produced human protein-enriched milk (2.4 gm per litre). It contains the human a-lactalbumin and is nutritionally more balanced product for human babies than natural cow-milk.
· Vaccine safety testing: Transgenic mice are used to test the safety of the polio vaccine. If it is found to be reliable, they can replace the use of monkeys to test the safety of batches of the vaccine.
· Chemical safety testing (toxicity testing): Transgenic animals are made that carry genes which make them more sensitive to toxic substances than non-transgenic animals. They are exposed to the toxic substances and the effects studied. It gives immediate results.
ETHICAL ISSUES
· Problem of unpredictable results: Genetic modification may cause unpredictable results when such organisms are introduced into the ecosystem.
Therefore, Indian Government has set up organizations like GEAC (Genetic Engineering Approval Committee), which make decisions about the validity of GM research and the safety of GM-organisms for public services.
· Problems of patent: Certain companies have got patents for products and technologies that make use of the genetic materials, plants etc that have been identified, developed and used by farmers and indigenous people of a specific country. E.g. Basmati rice, herbal medicines like turmeric, neem etc.
Basmati rice has unique aroma & flavour. India has 27 varieties of Basmati. In 1997, an American company got patent rights on Basmati rice through the US Patent and Trademark Office. This allowed the company to sell a ‘new’ variety of Basmati. This had actually been derived from Indian farmer’s varieties. Indian Basmati was crossed with semi-dwarf varieties and claimed as a novelty. Other people selling Basmati rice could be restricted by the patent.
· Biopiracy: Itis the use of bio-resources by multinational companies and other organizations without proper authorization from the countries and people concerned.
Most of the industrialized nations are poor in biodiversity and traditional knowledge. The developing and the underdeveloped world have rich biodiversity and traditional knowledge related to bio-resources.
It has to develop laws to prevent unauthorized exploitation of bio-resources and traditional knowledge.
Indian Parliament has cleared the second amendment of the Indian Patents Bill that takes such issues into consideration, including patent terms emergency provisions and research and development initiative.
