UPSC Biology Biotechnology : Principles And Processes Biotechnology: Principles and Processes

 Biotechnology: Principles and Processes

1.           Principles of Biotechnology

• Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.
• In this sense, making curd, bread or wine, which are all microbe-mediated processes, could also be thought as a form of biotechnology.
• However, it is used in a restricted sense today, to refer to such of those processes which use genetically modified organisms to achieve the same on a larger scale.
• Further, many other processes/techniques are also included under biotechnology. For example, in vitro fertilization leading to a 'test-tube9 baby, synthesising a gene and using it, developing a DNA vaccine or correcting a defective gene, are all part of biotechnology.
• The European Federation of Biotechnology (EFB) has given a definition of biotechnology that encompasses both traditional view and modem molecular biotechnology.
• We probably appreciate the advantages of sexual reproduction over asexual reproduction. The former provides opportunities for variations and formulation of unique combinations of genetic setup, some of which may be beneficial to the organism as well as the population. Asexual reproduction preserves the genetic information, while sexual reproduction permits variation.
• Traditional hybridisation procedures used in plant and animal breeding, very often lead to inclusion and multiplication of undesirable genes along with the desired genes.
• The techniques of genetic engineering which include creation of recombinant DNA, use of gene cloning and gene transfer, overcome this limitation and allows us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organism.                                
• The construction of the first recombinant DNA emerged from the possibility of linking a gene encoding antibiotic resistance with a native plasmid (autonomously replicating  circular extra-chromosomal DNA) of Salmonella typhimurium. Stanley Cohen and Herbert  Boyer accomplished this in 1972 by isolating the antibiotic resistance gene by cutting out a piece of DNA from a plasmid which was responsible for conferring antibiotic resistance.
• The cutting of DNA at specific locations became possible with the discovery of the so-called 'molecular scissors9- restriction enzymes. The cut piece of DNA was then linked with the plasmid DNA. These plasmid DNA act as vectors to transfer the piece of DNA attached to it.
• We probably know that mosquito acts as an insect vector to transfer the malarial parasite into human body. In the same way, a plasmid can be used as vector to deliver an alien piece of DNA into the host organism.
• The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends.
• This makes a new combination of circular autonomously replicating DNA created in vitro and is known as recombinant DNA. When this DNA is transferred into Escherichia coli, a bacterium closely related to Salmonella, it could replicate using the new host's DNA polymerase enzyme and make multiple copies. The ability to multiply copies of antibiotic resistance gene in E. coli was called cloning of antibiotic resistance gene in E. coli.
• There are three basic steps in genetically modifying an organism:
• identification of DNA with desirable genes;
• introduction of the identified DNA into the host;
• maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

2.           Tools of Reeombinant DNA Technology (Restriction Enzymes)

• In the year 1963, 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.
• The first restriction endonuclease-Hind II, whose functioning depended on a specific DNA nucleotide sequence was isolated and characterised five years later. It was found that Hind II always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs. This specific base sequence is known as the recognition sequence for Hind II.
• Besides Hind II, today we know7 more than 900 restriction enzymes that have been isolated from over 230 strains of bacteria each of which recognize different recognition sequences.
• Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two kinds; exonucleases and endonucleases. Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at specific positions within the DNA.
• 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 point's in their sugar-phosphate backbones. Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA.
• These are groups of letters that form the same words when read both forward and backward, e.g., "MALAYALAM". As against a word-palindrome where the same word is read in both directions, the palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.
• Restriction endonucleases are used in genetic engineering to form 'recombinant’ molecules of DNA, which are composed of DNA from different sources/genomes.
• When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of 'sticky-ends' and, these can be joined together (end-to-end) using DNA ligases.
• The cutting of DNA by restriction endonucleases results in the fragments of DNA. These fragments can be separated by a technique known as gel electrophoresis.
• Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix.
• Nowadays the most commonly used matrix is agarose which is 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 separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation.        
• The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.
• Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA. Bacteriophages because of their high number per cell, have very high copy numbers of their genome within the bacterial cells,         
• We have learnt the lesson of transferring genes into plants and animals from bacteria and viruses which have known this for ages - how to deliver genes to transform eukaryotic cells and force them to do what the bacteria or viruses want. For example, Agrobacterium tumifaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known  as 'T-DNA' to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen.                                    
• Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells. A better understanding of the art of delivering genes by pathogens in their eukaryotic hosts has generated knowledge to transform these tools of pathogens into useful vectors for delivering genes of interest to humans,                                
• Since DNA is a hydrophilic molecule, it cannot pass through cell membranes.
• In order to force bacteria to take up the plasmid, the bacterial cells must first be made 'competent' to take up DNA.                                
• This is done by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.                                                                    
• Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at
• \[42{}^\circ C\] (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.
• This is not the only way to introduce alien DNA into host cells. In a method known as micro-injection, recombinant DNA is directly injected into the nucleus of an animal cell.
• In another method, suitable for plants, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in a method known as biolistics or genegun.
• And the last method uses 'disarmed pathogen' vectors, which when allowed to infect the cell, transfer the recombinant DNA into the host.

3.           Biotechnological Applications in Agriculture

• Plants, bacteria, fungi and animals whose genes have been altered by manipulation are called Genetically Modified Organisms (GMO). GM plants have been useful in many ways.
• Genetic modification has :
• made crops more tolerant to abiotic stresses (cold, drought, salt, heat),
• reduced reliance on chemical pesticides (pest-resistant crops).
• helped to reduce post harvest losses.
• increased efficiency of mineral usage by plants (this prevents early exhaustion of fertility of soil).
• enhanced nutritional value of food, e.g.. Vitamin 'A5 enriched rice.
• In addition to these uses, GM has been used to create tailor-made plants to supply alternative resources to industries, in the form of starches, fuels and pharmaceuticals.
• In agriculture that you will study in detail are the production of pest resistant plants, which could decrease the amount of pesticide used.
• Bt toxin is produced by a bacterium called Bacillus thuringiensis (Bt for short). Bt toxin gene has been cloned from the bacteria and been expressed in plants to provide resistance to insects without the need for insecticides; in effect created a bio-pesticide. Examples are Bt cotton, Bt corn, rice, tomato, potato and soyabean etc.
• Some strains of Bacillus thuringiensis produce proteins that kill certain insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). Bacillus thuringiensis forms protein crystals during a particular phase of their growth. These crystals contain a toxic insecticidal protein.
• Why does this toxin not kill the Bacillus? Actually, the Bt toxin protein exist as inactive protoxins but once an insect ingest the inactive toxin, it is converted into an active form of toxin due to the alkaline pH of the gut which solubilise the crystals. The activated toxin binds to the surface of midgut epithelial cells and create pores that cause cell swelling and lysis and eventually cause death of the insect.
• Specific Bt toxin genes were isolated from Bacillus thuringiensis and incorporated into the several crop plants such as cotton. The choice of genes depends upon the crop and the targeted pest, as most Bt toxins are insect-group specific. The toxin is coded by a gene named cry. There are a number of them, for example, the proteins encoded by the genes cryIAc and cryIlAb control the cotton bollworms, that of crylAb controls corn borer.
• Several nematodes parasitise a wide variety of plants and animals including human beings. A nematode Meloidegyne incognitia infects the roots of tobacco plants and causes a great reduction in yield.
• A novel strategy was adopted to prevent this infestation which was based on the process of RNA interference (RNAi). RNAi takes place in all eukaryotic organisms as a method of cellular defense. This method involves silencing of a specific mRNA due to a complementary dsRNA molecule that binds to and prevents translation of the mRNA (silencing).
• The source of this complementary RNA could be from an infection by viruses having RNA genomes or mobile genetic elements (transposons) that replicate via an RNA intermediate.
• Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant. The introduction of DNA was such that it produced both sense and anti-sense RNA in the 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 the nematode. The consequence was that the parasite could not survive in a transgenic host expressing specific interfering RNA. The transgenic plant therefore got itself protected from the parasite.

4.           Biotechnological Applications in Medicine

• The recombinant DNA technological processes have made immense impact in the area of healthcare by enabling mass production of safe and more effective therapeutic drugs.
• Further, the recombinant therapeutics do 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 the world over. 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. What would a diabetic patient do if enough human-insulin was not available? One would have to isolate and use insulin from other animals.
• Insulin used for diabetes was earlier extracted from pancreas of slaughtered cattle and pigs. Insulin from an animal source, though caused some patients to develop allergy or other types of reactions to the foreign protein.
• Insulin consists of two short polypeptide chains: chain A and chain B, that are linked together by disulphide bridges. In mammals, including humans, insulin is synthesized as a pro-hormone (like a pro-enzyme, the pro-hormone also needs to be processed before it becomes a fully mature and functional hormone) which contains an extra stretch called the C peptide. This C peptide is not present in the mature insulin and is removed during maturation into insulin.
• The main challenge for production of insulin using rDNA techniques was getting insulin assembled into a mature form. In 1983, Eli Lilly an American company prepared two DNA sequences corresponding to A and B, chains of human insulin and introduced them in plasmids of E. coli to produce insulin chains. Chains A and B were produced separately, extracted and combined by creating disulfide bonds to form human insulin.

5.           Gene Therapy

• If a person is born with a hereditary disease, can a corrective therapy be taken for such a disease? Gene therapy is an attempt to do this. Gene therapy is a collection of methods that allows correction of a gene defect that has been diagnosed in a child/embryo.
• Here genes are inserted into a person's cells and tissues to treat a disease. Correction of a genetic defect involves delivery of a normal gene into the individual or embryo to take over the function of and compensate for the non-functional gene.
• The first clinical gene therapy was given in 1990 to a 4-year old girl with adenosine deaminase (ADA) deficiency. This enzyme is crucial for the immune system to function. The disorder is caused due to the deletion of the gene for adenosine deaminase.
• In some children ADA deficiency can be cured by bone marrow transplantation; in others it can be treated by enzyme replacement therapy, in which functional ADA. is given to the patient by injection. But the problem with both of these approaches that they are not completely curative.
• As a first step towards gene therapy, lymphocytes from the blood of the patient are grown in a culture outside the body. A functional ADA cDNA (using a retroviral vector) is then introduced into these lymphocytes, which are subsequently returned to the patient.
• However, as these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. However, if the gene isolate from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure.

6.           Molecular Diagnosis

• For effective treatment of a disease, early diagnosis and understanding its pathophysiology is very important.
• Using conventional methods of diagnosis (serum and urine analysis, etc.) early detection is not possible. Recombinant DNA technology, Polymerase Chain Reaction (PCR) and Enzyme Linked Immuno-sorbent Assay (ELISA) are some of the techniques that serve the purpose of early diagnosis.
• Presence of a pathogen (bacteria, viruses, etc.) is normally suspected only when the pathogen has produced a disease symptom. By this time the concentration of pathogen is already very high in the body. However, very low concentration of a bacteria or virus (at a time when the symptoms of the disease are not yet visible) can be detected by amplification of their nucleic acid by PCR.
• PCR is now routinely used to detect HIV in suspected AIDS patients. It is being used to detect mutations in genes in suspected cancer patients too. It is a powerful techqnique 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 complementarity with the mutatedgene.
• 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 synthesised against the pathogen.

7.           Transgenic Animals

• Animals that have had their DNA manipulated to possess and express an extra (foreign) gene are known as transgenic animals. Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, although over 95 per cent of all existing transgenic animals are mice. Why are these animals being produced?
• Transgenic animals can be specifically designed to allow the study of how genes are regulated, and how they affect the normal functions of the body and its development, e.g., study of complex factors involved in growth such as insulin-like growth factor. By introducing genes from other species that alter the formation of this factor and studying the biological effects that result, information is obtained about the biological role of the factor in the body.
• Many transgenic animals are designed to increase our understanding of how genes contribute to the development of disease. These are specially made to serve as models for human diseases so that investigation of new treatments for diseases is made possible. Today transgenic models exist for many human diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's.
• Medicines required to treat certain human diseases can contain biological products, but such products are often expensive to make Transgenic animals that produce useful biological products can be created by the introduction of the portion of DNA (or genes) which codes for a particular product such as human protein (
• \[\alpha\]
• -1-antitrypsin) used to treat emphysema. Similar attempts are being made for treatment of phenylketonuria (PKU) and cystic fibrosis. In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk (\[2.4\]grams per litre). The milk contained the human alpha-lactalbumin and was nutritionally a more balanced product for human babies than natural cow-milk.
• Transgenic mice are being developed for use in testing the safety of vaccines before they are used on humans. Transgenic mice are being used to test the safety of the polio vaccine. If successful and found to be reliable, they could replace the use of monkeys to test the safety of batches of the vaccine.
• Chemical Safety Testing : This is known as toxicity/safety testing. The procedure is the same as that used for testing toxicity of drugs. Transgenic animals are made that carry genes which make them more sensitive to toxic substances than non-transgenic animals. They are then exposed to the toxic substances and the effects studied. Toxicity testing in such animals will allow us to obtain results in less time.