Biotechnology and its Applications

Chapter 9 gave you the tool kit: restriction enzymes, vectors, competent hosts, PCR, bioreactors and downstream processing. This chapter answers the question every student should ask at the end of that one: what has all this actually produced? NCERT's answer is that biotechnology deals with the industrial-scale production of biopharmaceuticals and biologicals using genetically modified microbes, fungi, plants and animals. The applications it lists are worth remembering as a set, because a 1-mark question can ask for any four of them: therapeutics, diagnostics, genetically modified crops for agriculture, processed food, bioremediation, waste treatment and energy production.

NCERT also names three critical research areas on which all of this rests, and they turn up as a 3-mark question more often than you would expect:

1. Providing the best catalyst in the form of an improved organism, usually a microbe, or a pure enzyme.
2. Creating optimal conditions through engineering for that catalyst to act.
3. Downstream processing technologies to purify the protein or organic compound.

Keep the order and the wording; the examiner is looking for the words catalyst, optimal conditions and downstream processing.

The chapter then walks through four fields in a fixed order, and that order is how you should revise it: agriculture (10.1), medicine (10.2), transgenic animals (10.3) and ethical issues (10.4). Within agriculture there are two worked examples, Bt cotton and RNAi-protected tobacco. Within medicine there are three, genetically engineered insulin, gene therapy for ADA deficiency and molecular diagnosis. Almost every board question from this chapter is one of these five stories told in full, or a single fact pulled out of one of them. If you can narrate each story in the right sequence with the right names, the chapter is done.

One more thing about the CBSE syllabus line for this chapter: it lists human insulin and vaccine production, stem cell technology, gene therapy, Bt crops, transgenic animals, biosafety, biopiracy and patents. The NCERT text does not have a separate section on stem cells or vaccines, so questions on those stay at the level of a sentence: the hepatitis B vaccine is made in yeast by rDNA technology (you met this in Chapter 7), and stem cells are unspecialised cells that can be coaxed to form the cell types a patient has lost.

NCERT opens the agriculture section with a choice. To increase food production there are three options: agro-chemical based agriculture, organic agriculture, and genetically engineered crop-based agriculture. The Green Revolution succeeded in tripling the food supply, but that is still not enough to feed the growing human population, and its gains came only partly from improved crop varieties; mainly they came from better management practices and the use of agrochemicals (fertilisers and pesticides). Two problems follow. Agrochemicals are often too expensive for farmers in the developing world, and conventional breeding cannot push yields any further. That is the case for the third option.

A genetically modified organism (GMO) is a plant, bacterium, fungus or animal whose genes have been altered by manipulation. This is the definition the examiner wants for 'What is a GMO?'. NCERT then gives the list of what genetic modification has done for plants, and this list is asked directly as 'advantages of GM crops' or 'arguments in favour of GM crops':

1. Made crops more tolerant to abiotic stresses: cold, drought, salt, heat.
2. Reduced reliance on chemical pesticides, through pest-resistant crops.
3. Helped to reduce post-harvest losses.
4. Increased the efficiency of mineral usage by plants, which prevents early exhaustion of soil fertility.
5. Enhanced the nutritional value of food, for example vitamin A-enriched rice (golden rice).

NCERT adds a sixth use in the same paragraph: GM has been used to create tailor-made plants that supply alternative resources to industries in the form of starches, fuels and pharmaceuticals. A 5-mark question on 'potential applications of GM plants' should include this along with the five above.

The disadvantages are not spelt out in this section but they are asked (NCERT exercise 3 says 'compare and contrast'). Draw them from the ethics section and general knowledge: unpredictable effects when a GMO enters an ecosystem, gene flow to wild relatives, possible allergenicity of the new protein, evolution of resistance in the target pest, loss of biodiversity if a few GM varieties dominate, dependence of farmers on seed companies, and patent disputes.

This is the most-asked story in the chapter. Tell it in this order.

The bacterium. Bacillus thuringiensis (Bt for short) is a soil bacterium that produces proteins that kill certain insects: lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). During a particular phase of its growth the bacterium forms protein crystals, and these crystals contain a toxic insecticidal protein.

Why the bacterium is not killed by its own toxin. The Bt toxin exists inside the bacterium as an inactive protoxin. Nothing happens until an insect ingests it. In the insect's gut the alkaline pH solubilises the crystals and converts the protoxin into the active toxin. The activated toxin binds to the surface of the midgut epithelial cells and creates pores. The cells swell and lyse, and the insect dies. Marking schemes for 'why do lepidopterans die on Bt cotton' reward exactly these steps: ingestion, alkaline gut, activation, binding to midgut epithelium, pores, swelling and lysis, death.

The genes. Specific Bt toxin genes were isolated from Bacillus thuringiensis and incorporated into several crop plants such as cotton. The genes are named cry (from crystal), and there are many of them. The choice of gene depends on the crop and the target pest, because most Bt toxins are insect-group specific. Learn three: proteins coded by cryIAc and cryIIAb control the cotton bollworm; the protein coded by cryIAb controls the corn borer. The 2019 board paper asked for the two bollworm genes as a 1-mark question, and students who wrote cryIAb lost the mark.

What the plant then does. A Bt cotton plant carries the cry gene and therefore makes the crystal protein in its own tissues. A bollworm larva that bites the boll eats the protoxin, its alkaline gut activates it, and it dies. NCERT's Figure 10.1 contrasts a cotton boll destroyed by bollworms with a fully mature, healthy boll; the point of the figure is that the transgenic plant no longer needs chemical insecticide against that pest.

Two naming details that cost marks: the gene is written in lower-case italics, cry, and the protein is Cry with a capital C. Also, Bt toxin is a protein, not a chemical insecticide, and it is specific, so it does not harm insects that lack the alkaline gut and the receptors.

The second agricultural story is about a worm, not an insect. Several nematodes parasitise a wide variety of plants and animals, humans included. One of them, Meloidogyne incognita (NCERT prints it as Meloidegyne incognitia; either spelling is accepted), infects the roots of tobacco plants and causes a large reduction in yield. The strategy used against it is based on RNA interference (RNAi).

What RNAi is. RNAi takes place in all eukaryotic organisms as a method of cellular defence. It involves silencing of a specific mRNA by a complementary double-stranded RNA (dsRNA) that binds to the mRNA and prevents its translation. That is the whole definition and it is the 2-mark answer to 'What is RNAi?'. Where does a cell naturally get such complementary RNA? NCERT gives two sources: an infection by viruses with RNA genomes, or mobile genetic elements (transposons) that replicate through an RNA intermediate. 'How does dsRNA gain entry into a eukaryotic cell?' has been asked in exactly these words.

The strategy, in sequence. (1) Nematode-specific genes were introduced into the host tobacco plant using Agrobacterium vectors. (2) The DNA was introduced in such a way that it produced both sense and antisense RNA in the host cells. (3) These two RNAs, being complementary, base-paired to form dsRNA. (4) The dsRNA initiated RNAi and silenced the specific mRNA of the nematode. (5) The parasite, unable to make that essential protein, could not survive in a transgenic host expressing the interfering RNA, so the plant protected itself. The 2018 paper asked for this 'in correct sequence', which tells you what the marking scheme looks like: one mark for each numbered step, and steps out of order lose marks.

NCERT's Figure 10.2 shows three root systems side by side: (a) the roots of a typical healthy plant, (b) the roots of a nematode-infested plant, with knots and stunted growth, and (c) the roots of the transgenic plant, healthy again. If you are asked to draw it, three simple root sketches with those three captions are enough.

Notice the logic that links this to Bt cotton. In both cases the plant is given a gene and makes a product in its own tissues; in Bt the product is a protein that kills the pest, in RNAi the product is an RNA that switches off a gene in the pest. Questions often ask you to name 'the strategy' (RNAi) and 'the vector' (Agrobacterium) separately, so keep both words ready.

The medicine section begins with a claim that is worth quoting in an answer: recombinant DNA technology has had an immense impact on healthcare by enabling the mass production of safe and more effective therapeutic drugs. The important adjective is safe. Recombinant therapeutics do not induce unwanted immunological responses, which is common when the same product is isolated from a non-human source. This one sentence is the reason recombinant insulin replaced animal insulin, and it is the reason recombinant human growth hormone replaced hormone extracted from cadaver pituitaries; keep it in mind for every question on 'why recombinant?'.

NCERT gives two numbers and both have been asked as 1-mark fill-ups: at present about 30 recombinant therapeutics have been approved for human use the world over, and in India 12 of these are being marketed. Recombinant products in this category include insulin, growth hormone, erythropoietin, interferons, clotting factor VIII and the hepatitis B vaccine; NCERT names only insulin in detail, so the others are for context and for the 'vaccine production' line of the syllabus.

The section then breaks into three subsections, and you should be able to say in one line what each is about before you learn the details:

• 10.2.1 Genetically engineered insulin: how a human hormone is made in a bacterium and why the pro-hormone structure was the hard part.
• 10.2.2 Gene therapy: how a missing gene can be supplied to a patient's cells, with ADA deficiency as the first clinical case.
• 10.2.3 Molecular diagnosis: how PCR, DNA probes and ELISA detect a disease before symptoms appear.

A common 3-mark question strings these together: 'Mention three applications of biotechnology in medicine, with one example each.' The answer is therapeutics (recombinant insulin), gene therapy (ADA deficiency) and diagnostics (PCR for HIV, ELISA). Add the hepatitis B vaccine from yeast if a fourth is asked. The next three sections give each story in full.

The problem. Adult-onset diabetes can be managed by taking insulin at regular intervals. Insulin was earlier extracted from the pancreas of slaughtered cattle and pigs. This animal insulin worked, but it caused some patients to develop allergy or other reactions to the foreign protein. That is the 2-mark answer to 'why is animal insulin no longer used'; a slaughter-based supply also could not keep pace with demand, which you may add.

The structure. Insulin consists of two short polypeptide chains, chain A and chain B, linked together by disulphide bridges. (Chain A has 21 amino acids and chain B has 30; NCERT does not give the numbers but they help you draw the figure to scale.) In mammals, including humans, insulin is synthesised as a pro-hormone, which, like a pro-enzyme, must be processed before it becomes a fully mature and functional hormone. Pro-insulin contains an extra stretch called the C peptide that connects chain A to chain B. The C peptide is not present in mature insulin; it is removed during maturation. So the 'chemical change that pro-insulin undergoes to become mature insulin' (asked in 2018) is the removal of the C peptide, and the 'role of the C peptide' (asked in 2014) is that it holds the A and B chains together in the pro-hormone and is cut out on maturation.

The challenge. A bacterium has no machinery to cut the C peptide out of pro-insulin. So the main challenge in making insulin by rDNA technology was getting insulin assembled into its mature form.

The solution. In 1983, Eli Lilly, an American company, prepared two DNA sequences corresponding to the A and B chains of human insulin and introduced them into plasmids of E. coli to produce the insulin chains. Chains A and B were produced separately, extracted, and combined by creating disulphide bonds to form human insulin. The company went round the C-peptide problem by never making pro-insulin at all. This recombinant insulin, sold as humulin, is identical to the human hormone and does not provoke the reactions that animal insulin did.

The difference examiners ask for. Insulin made by the human pancreas is first synthesised as pro-insulin with three parts (A, B and C peptide) and matured by removing C. Insulin made by Eli Lilly is assembled directly from separately made A and B chains; no C peptide is ever involved. Write both halves of this contrast for a 2-mark 'how does it differ' question.

NCERT's Figure 10.3, 'maturation of pro-insulin into insulin (simplified)', is a frequent diagram question and is described in the diagrams section below.

Definition. Gene therapy is a collection of methods that allows correction of a gene defect that has been diagnosed in a child or embryo. Genes are inserted into a person's cells and tissues to treat a disease. The correction involves the delivery of a normal gene into the individual or embryo to take over the function of, and compensate for, the non-functional gene. Write the definition with the word 'compensate'; that is NCERT's key idea. The defective gene is not repaired or removed, it is supplemented.

The first case. The first clinical gene therapy was given in 1990 to a 4-year-old girl with adenosine deaminase (ADA) deficiency. ADA is an enzyme crucial for the immune system to function; without it lymphocytes cannot survive and the child has severe combined immunodeficiency (SCID). The disorder is caused by the deletion of the gene for adenosine deaminase. When asked 'the cause and the body system affected', the answer is gene deletion and the immune system.

Two conventional treatments, and why neither is enough. In some children ADA deficiency can be cured by bone marrow transplantation. In others it can be treated by enzyme replacement therapy (ERT), in which functional ADA is given to the patient by injection. The problem with both approaches is that they are not completely curative: a matched donor is not always available, and injected enzyme is used up, so the injections must be repeated for life. The 2016 paper asked for ERT and two of its disadvantages, and the marking scheme wanted 'not completely curative' and 'requires periodic injections'.

The gene therapy protocol, in sequence. (1) Lymphocytes from the patient's blood are grown in culture outside the body. (2) A functional ADA cDNA is introduced into these lymphocytes using a retroviral vector. (3) The engineered lymphocytes are returned to the patient. (4) Because lymphocytes are not immortal, the patient needs periodic infusion of such genetically engineered lymphocytes. Note two things students get wrong: it is cDNA (made from ADA mRNA by reverse transcriptase, so it has no introns and can be expressed in the target cell) and the vector is a retrovirus, not a plasmid.

The permanent cure. If the gene isolated from marrow cells producing ADA is introduced into cells at an early embryonic stage, it could be a permanent cure, because every cell of the body, including the stem cells that keep producing lymphocytes, would then carry the working gene. This is the standard 1-mark 'suggest a permanent cure' answer.

Why it matters. For effective treatment of a disease, early diagnosis and an understanding of its pathophysiology are very important. Conventional methods (serum and urine analysis and the like) cannot detect a disease early, because the presence of a pathogen is normally suspected only when it has already produced a symptom, and by then its concentration in the body is already very high. Three techniques serve the purpose of early diagnosis: recombinant DNA technology, PCR and ELISA. 'Why is molecular diagnosis preferred over conventional methods?' (asked in 2016) wants exactly this: early detection, before symptoms, when pathogen numbers are still very low.

PCR. A very low concentration of a bacterium or virus, at a time when symptoms are not yet visible, can be detected by amplification of its nucleic acid by PCR. PCR is now routinely used to detect HIV in suspected AIDS patients. It is also used to detect mutations in genes in suspected cancer patients, and it is a powerful technique for identifying many other genetic disorders. Give both examples, HIV and cancer, when asked for the uses of PCR in diagnosis.

The radioactive probe. A single-stranded DNA or RNA tagged with a radioactive molecule is called a probe. It is allowed to hybridise to its complementary DNA in a clone of cells, and the result is read by autoradiography. The logic of detecting a mutation is the part students must state clearly: the probe is made complementary to the normal gene, so the clone carrying the mutated gene does not appear on the photographic film, because the probe has no complementarity with the mutated sequence and does not bind. Normal cells light up; mutant cells stay dark.

ELISA. The Enzyme-Linked Immuno-Sorbent Assay is based on the principle of antigen–antibody interaction. Infection by a pathogen can be detected in two ways: by the presence of antigens (proteins, glycoproteins and so on) of the pathogen, or by detecting the antibodies that the body has synthesised against the pathogen. In Chapter 7 you learnt that ELISA is the usual screening test for HIV, and the two chapters are often linked in one question: PCR detects the viral nucleic acid, ELISA detects the viral antigen or the antibody to it.

A neat way to keep the three straight for a 3-mark question: PCR detects the pathogen's nucleic acid; a probe detects a specific gene sequence (normal versus mutant); ELISA detects a protein (antigen or antibody). One line each with an example, and the marks are yours.

Definition. 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. The definition and the 95 per cent figure are both 1-mark regulars.

NCERT asks why such animals are made and gives five purposes. Learn the heading of each, then one example under it; a 5-mark question wants all five, a 3-mark question wants any three with examples.

1. Normal physiology and development. Transgenic animals are designed to allow the study of how genes are regulated and how they affect the normal functions of the body and its development. NCERT's example is the insulin-like growth factor: by introducing genes that alter its formation and studying the biological effects, we learn about the biological role of the factor.
2. Study of disease. Many transgenic animals are designed to increase our understanding of how genes contribute to the development of disease. They serve as models for human diseases so that new treatments can be investigated. Transgenic models exist for cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's.
3. Biological products. Medicines for certain human diseases contain biological products that are expensive to make. A transgenic animal that produces a useful product can be created by introducing the DNA (gene) that codes for it. The example is the human protein α-1-antitrypsin, used to treat emphysema; similar attempts are being made for 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 human α-lactalbumin and was a nutritionally more balanced product for human babies than natural cow milk. 'How is Rosie different from a normal cow?' has been asked twice; the answer is the α-lactalbumin, the quantity and the reason it matters.
4. Vaccine safety. Transgenic mice are being developed to test the safety of vaccines before they are used on humans, and are already being used to test the safety of the polio vaccine. If found reliable, they could replace the use of monkeys for testing batches of the vaccine.
5. Chemical safety testing. Also called toxicity or safety testing, this follows the same procedure as drug testing. Transgenic animals are made that carry genes making them more sensitive to toxic substances than non-transgenic animals. They are exposed to the substance and the effects are studied; toxicity testing in such animals gives results in less time.

A trap in this section: Rosie's milk contained human α-lactalbumin, not α-1-antitrypsin. The two 'alpha' proteins belong to two different sentences of NCERT and examiners deliberately test whether you have kept them apart.

NCERT starts this section with a warning: the manipulation of living organisms by the human race cannot go on any further without regulation. Some ethical standards are needed to evaluate the morality of all human activities that might help or harm living organisms. Beyond morality, the biological consequences also matter: genetic modification of organisms can have unpredictable results when such organisms are introduced into the ecosystem. That sentence is the standard answer to 'why is there concern about releasing GMOs'.

GEAC. To deal with this, the Indian Government has set up organisations such as the Genetic Engineering Approval Committee (GEAC). Its job, in NCERT's words, is to make decisions regarding (i) the validity of GM research and (ii) the safety of introducing GM organisms for public services. Those two clauses are the two 'objectives of GEAC' asked in 2015. For your information, the body was renamed the Genetic Engineering Appraisal Committee in 2010 and sits under the Ministry of Environment, Forest and Climate Change; write the NCERT expansion in the exam, and add the current name in brackets if you wish. GEAC's approval was needed, for example, before Bt cotton could be grown commercially in India in 2002.

Patents on living material. The modification and use of living organisms for public services (as food and medicine sources, for example) has also created problems with patents. There is growing public anger that companies are being granted patents for products and technologies that make use of genetic materials, plants and other biological resources that have long been identified, developed and used by farmers and indigenous people of a specific region or country. This is the seed of the biopiracy idea developed in the next section.

Two more points that can complete a 5-mark answer on biosafety and ethics. First, the Cartagena Protocol on Biosafety (2000, under the Convention on Biological Diversity) governs the international movement of living modified organisms; India is a party. Second, ethical questions about transgenic animals include the welfare of the animals themselves, and questions about gene therapy include whether changes should ever be made to the embryo (the germ line), since those would be inherited. NCERT does not detail these, so keep them to a sentence each.

The Basmati story. Rice has been grown in Asia for thousands of years, and India alone has an estimated 200,000 varieties; the diversity of rice in India is one of the richest in the world. Basmati is distinct for its unique flavour and aroma, there are 27 documented varieties of Basmati grown in India, and it appears in ancient texts, folklore and poetry. In 1997, an American company (RiceTec, though NCERT does not name it) obtained patent rights on Basmati rice through the US Patent and Trademark Office. This allowed the company to sell a 'new' variety of Basmati in the US and abroad. That 'new' variety had actually been derived from Indian farmers' varieties: Indian Basmati was crossed with semi-dwarf varieties and claimed as an invention or novelty. The patent extended to functional equivalents, which meant that other people selling Basmati rice could be restricted by it. Several attempts have also been made to patent uses, products and processes based on Indian traditional herbal medicines, for example turmeric and neem. NCERT's conclusion is a sentence you can quote: if we are not vigilant and do not immediately counter such patent applications, other countries or individuals may encash on our rich legacy and we cannot do anything about it.

Definition of biopiracy. Biopiracy is the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concerned and without compensatory payment. Write all three parts: who, without authorisation, without compensation.

The North–South imbalance. Most industrialised nations are rich financially but poor in biodiversity and traditional knowledge. The developing and underdeveloped world is the reverse: rich in biodiversity and in traditional knowledge related to bio-resources. Traditional knowledge can be exploited to develop modern applications and can save time, effort and expenditure during commercialisation, which is exactly why it is worth taking. There has been a growing realisation of the injustice, inadequate compensation and benefit sharing between developed and developing countries.

What has been done. Some nations are developing laws to prevent such unauthorised exploitation of their bio-resources and traditional knowledge. The Indian Parliament has cleared the second amendment of the Indian Patents Bill, which takes such issues into consideration, including patent terms, emergency provisions and research and development initiative. 'Biopiracy should be prevented; state why and how' (2011) wants the definition, the injustice to the source community, and the Patents Bill amendment as the 'how'. India's Biological Diversity Act, 2002 and the Traditional Knowledge Digital Library are further measures you can mention; the neem and turmeric patents were in fact revoked after challenges backed by Indian institutions.