Ch.10 MICROBES IN HUMAN WELFARE IMPORTANT CONCEPTS AND DEFINNITIONS-
Ch.10 MICROBES IN HUMAN WELFARE IMPORTANT CONCEPTS AND DEFINNITIONS1. Fermentors – Production of beverages and antibiotics on an industrial scale, requires microbes in very large vessels called fermentors . growing 2. Antibiotics- Antibiotics are chemical substances, which are produced by some microbes and can kill or retard the growth of other (disease-causing) microbes. 3. Primary treatment : The treatment step of sewage for physical removal of particles – large and small – from the sewage through filtration and sedimentation , is called . Primary treatment . 4. Secondary treatment or Biological treatment : After primary treatment .the primary effluent is passed into large aeration tanks where it is constantly agitated mechanically and air is pumped associated with fungal filaments to form mesh like structures). 5. BOD (biochemical oxygen demand) -BOD refers to the amount of the oxygen that would be consumed if all the organic matter in one liter of water were oxidised by bacteria 6. Activated sludge- Once the BOD of sewage or waste water is reduced significantly, the effluent is then passed into a settling tank where the bacterial ‘flocs’ are allowed to sediment. This sediment is called activated sludge. 7. Anaerobic sludge digesters- A small part of the activated sludge is pumped back into the aeration tank to serve as the inoculum. The remaining major part of the sludge is pumped into large tanks called anaerobic sludge digesters.where anaerobica bacteria and the fungi digest the sludge. 8. Methanogens- Certain bacteria, which grow anaerobically on cellulosic material, produce large amount of methane along with CO2 and H2. These bacteria are collectively called methanogens, 9. Biocontrol refers to the use of biological methods (using biological agents for natural predation ) for controlling plant diseases and pests , rather than using chemicals . 10. Biofertilisers- In agriculture Biofertilisers are organisms that enrich the nutrient quality of the soil. 11. Organic farming –Agriculture by using only biofertilisers is called organic farming because there are problems of pollution , associated with the overuse of chemical fertilizers. The main sources of biofertilisers are bacteria, fungi and cyanobacteria. 12. Mycorrhiza- Fungal symbiotic association with plants which helps plants to absorb phosphorus from soil , is called mycorrhiza. Many members of the genus Glomus form mycorrhiza.. 13. Cyanobacteria -Cyanobacteria are autotrophic microbes widely distributed in aquatic and terrestrial environments many of which can fix atmospheric nitrogen, e.g. Anabaena, Nostoc, Oscillatoria, etc. IDENTICAL TERMS1. Fermented Beverages- Wine and beer are produced without distillation after fermentation so they are called as fermented beverages. Distillation Beverages- Whisky, brandy and rum are produced by distillation of the fermented broth that’s why they are called “Distillation Beverages.” 2. Acetobactor aceti- A bacterium which produces acetic acid commercially. Lactobacillus- A bacterium which grows in milk and convert it into curd by producing lactic acid. 3. Primary Sludge- After primary treatment all solids that settle down like soil, small pebbles, settled debris etc.is called primary sludge. Activated Sludge- During secondary treatment the ‘flocs’ (masses of bacteria associated with fungal filaments to form mesh like structures) are allowed to sediment. This sediment is called activated sludge. 4. Endomycorrhiza- Intimated fungal association with certain woody plants is endomycorrhiza. Ectomycorrhiza- superficial fungal association with certain woody plants is endomycorrhiza. 5. Biocontrol - A method of controlling pests that relies on natural predation rather than introduced chemicals. Biofertilizers- Biofertilisers are organisms that enrich the nutrient quality of the soil. IMPORTANT POINTS, PRINCIPLES AND THEORYMICROBES IN HOUSEHOLD PRODUCTSMicro-organisms such as Lactobacillus and others commonly called lactic acid bacteria (LAB) grow in milk and convert it to curd. LAB produce acids that coagulate and partially digest the milk proteins. It also improves its nutritional quality by increasing vitamin B12. In our stomach too, the LAB play very beneficial role in checking diseasecausing microbes. The dosa and idli is also fermented by bacteria. The dough, which is used for making bread, is fermented using baker’s yeast (Saccharomyces cerevisiae). A number of traditional drinks and foods are also made by fermentation by the microbes. ‘Toddy’, a traditional drink of some parts of southern India is made by fermenting sap from palms. Microbes are also used to ferment fish, soyabean and bamboo shoots to make foods. Cheese, is one of the oldest food items in which microbes were used. The large holes in ‘Swiss cheese’ are due to production of a large amount of CO2 by a bacterium named Propionibacterium sharmanii. The ‘Roquefort cheese’ are ripened by growing a specific fungi on them, which gives them a particular flavour. MICROBES IN INDUSTRIAL PRODUCTSIin industry, microbes are used to synthesise a number of products valuable to human beings. Beverages and antibiotics are some examples. Production on an industrial scale, requires growing microbes in very large vessels called fermentors . Fermented BeveragesMicrobes like yeast are used for the production of beverages like wine, beer, whisky, brandy or rum. The same yeast Saccharomyces cerevisiae used for bread-making and commonly called brewer’s yeast, is used for fermenting malted cereals and fruit juices, to produce ethanol. Wine and beer are produced without distillation whereas whisky, brandy and rum are produced by distillation of the fermented broth. AntibioticsAnti is a Greek word that means ‘against’, and bio means ‘life’, together they mean ‘against life’ (in the context of disease causing organisms); whereas with reference to human beings, they are ‘pro life’ and not against. Antibiotics are chemical substances, which are produced by some microbes and can kill or retard the growth of other (disease-causing) microbes. Alexander Fleming while working on Staphylococci bacteria, once observed a mould growing in one of his unwashed culture plates around which Staphylococci could not grow. He found out that it was due to a chemical produced by the mould and he named it Penicillin after the mould Penicillium notatum. Its full potential as an effective antibiotic was established by Ernest Chain and Howard Florey. This antibiotic was extensively used to treat American soldiers wounded in World War II. Fleming, Chain and Florey were awarded the Nobel Prize in 1945, for this discovery. Antibiotics have greatly improved our capacity to treat deadly diseases such as plague, whooping cough (kali khansi ), diphtheria (gal ghotu) and leprosy (kusht rog), which used to kill millions all over the globe. Today, we cannot imagine a world without antibiotics. Chemicals, Enzymes and other Bioactive MoleculesAspergillus niger (a fungus) produces citric acid, Acetobacter aceti (a bacterium) produces acetic acid; Clostridium butylicum (a bacterium) produces butyric acid Lactobacillus (a bacterium) produces lactic acid. Yeast (Saccharomyces cerevisiae) is used for commercial production of ethanol. Enzymes like Lipases are used in detergent formulations and are helpful in removing oily stains from the laundry. The bottled juices are clarified by the use of pectinases and proteases. Streptokinase produced by the bacterium Streptococcus and modified by genetic engineering is used as a ‘clot buster’ for removing clots from the blood vessels of patients who have undergone myocardial infraction leading to heart attack. Cyclosporin A, that is used as an immunosuppressive agent in organ-transplant patients, is produced by the fungus Trichoderma polysporum. Statins produced by the yeast Monascus purpureus have been commercialised as blood-cholesterol lowering agents. It acts by competitively inhibiting the enzyme responsible for synthesis of cholesterol. MICROBES IN SEWAGE TREATMENTThe municipal waste-water including human excreta is also called sewage. It contains large amounts of organic matter and microbes. Many of which are pathogenic. This cannot be discharged into natural water bodies like rivers and streams directly .Before disposal, hence, sewage is treated in sewage treatment plants (STPs) to make it less polluting. Treatment of waste water is done by the heterotrophic microbes naturally present in the sewage. This treatment is carried out in two stages: Primary treatment : These treatment steps basically involve physical removal of particles – large and small – from the sewage through filtration and sedimentation. These are removed in stages; initially, floating debris is removed by sequential filtration. Then the grit (soil and small pebbles) are removed by sedimentation. All solids that settle form the primary sludge, and the supernatant forms the effluent. The effluent from the primary settling tank is taken for secondary treatment. Secondary treatment or Biological treatment : The primary effluent is passed into large aeration tanks ,where it is constantly agitated mechanically and air is pumped into it. This allows vigorous growth of useful aerobic microbes into flocs (masses of bacteria associated with fungal filaments to form mesh like structures). While growing, these microbes consume the major part of the organic matter in the effluent. This significantly reduces the BOD (biochemical oxygen demand) of the effluent. BOD refers to the amount of the oxygen that would be consumed if all the organic matter in one liter of water were oxidised by bacteria. The sewagewater is treated till the BOD is reduced. The BOD test measures the rate of uptake of oxygen by micro-organisms in a sample of water and thus, indirectly, BOD is a measure of the organic matter present in the water. The greater the BOD of waste water, more is its polluting potential. Once the BOD of sewage or waste water is reduced significantly, the effluent is then passed into a settling tank where the bacterial ‘flocs’ are allowed to sediment. This sediment is called activated sludge. A small part of the activated sludge is pumped back into the aeration tank to serve as the inoculum. The remaining major part of the sludge is pumped into large tanks called anaerobic sludge digesters. Here, other kinds of bacteria, which grow anaerobically, digest the bacteria and the fungi in the sludge. During this digestion, bacteria produce a mixture of gases such as methane, hydrogen sulphide and carbon dioxide. These gases form biogas and can be used as source of energy as it is inflammable. MICROBES IN PRODUCTION OF BIOGASBiogas is a mixture of gases (containing predominantly methane) produced by the microbes and which may be used as fuel. Certain bacteria, which grow anaerobically on cellulosic material, produce large amount of methane along with CO2 and H2. These bacteria are collectively called methanogens, and one such common bacterium is Methanobacterium. These bacteria are found in the anaerobic sludge during sewage treatment. These bacteria are also present in the rumen (a part of stomach) of cattle. A lot of cellulosic material present in the food of cattle is also present in the rumen. In rumen, these bacteria help in the breakdown of cellulose and play an important role in the nutrition of cattle. Thus, the excreta (dung) of cattle, commonly called gobar, is rich in these bacteria. Dung can be used for generation of biogas, commonly called gobar gas. A TYPICAL BIOGAS PLANT The biogas plant consists of a concrete tank (10-15 feet deep) in which bio-wastes are collected and a slurry of dung is fed. A floating cover is placed over the slurry, which keeps on rising as the gas is produced in the tank due to the microbial activity. The biogas plant has an outlet, which is connected to a pipe to supply biogas to nearby houses. The spent slurry is removed through another outlet and may be used as fertiliser. Cattle dung is available in large quantities in rural areas where cattle are used for a variety of purposes. So biogas plants are more after build in rural areas. The biogas thus produced is used for cooking and lighting. The technology of biogas production was developed in India mainly due to the efforts of Indian Agricultural Research Institute (IARI) and Khadi and Village Industries Commission (KVIC). MICROBES AS BIOCONTROL AGENTSBiocontrol refers to the use of biological methods for controlling plant diseases and pests. Use of chemicals like insecticides and pesticides causes great harm. These chemicals are toxic and extremely harmful, to human beings and animals alike, and have been polluting our environment (soil, ground water), fruits, vegetables and crop plants. Our soil is also polluted through use of weedicides to remove weeds. Biological control of pests and diseases: It is a method of controlling pests that relies on natural predation rather than introduced chemicals. A key belief of the organic farmer is that biodiversity furthers health. The organic farmer works to create a system where the insects that are sometimes called pests are not eradicated, but instead are kept at manageable levels by a complex system of checks and balances within a living and vibrant ecosystem. This is a holistic approach that seeks to develop an understanding of the webs of interaction between the myriad of organisms that constitute the field fauna and flora. The very familiar beetle with red and black markings – the Ladybird, and Dragonflies are useful to get rid of aphids and mosquitoes, respectively. An example of microbial biocontrol agents that can be introduced in order to control butterfly caterpillars is the bacteria Bacillus thuringiensis (often written as Bt ). These are available in sachets as dried spores which are mixed with water and sprayed onto vulnerable plants such as brassicas and fruit trees, where these are eaten by the insect larvae. In the gut of the larvae, the toxin is released and the larvae get killed. A biological control being developed for use in the treatment of plant disease is the fungus Trichoderma. Trichoderma species are free-living fungi that are very common in the root ecosystems. Baculoviruses (genus Nucleopolyhedrovirus) are pathogens that attack insects and other arthropods. These viruses are excellent candidates for species-specific, narrow spectrum insecticidal applications. The beneficial insects are being conserved to aid in an overall integrated pest management (IPM) programme, or when an ecologically sensitive area is being treated. MICROBES AS BIOFERTILISERSBiofertilisers are organisms that enrich the nutrient quality of the soil. The main sources of biofertilisers are bacteria, fungi and cyanobacteria. The nodules on the roots of leguminous plants formed by the symbiotic association of Rhizobium. These bacteria fix atmospheric nitrogen into organic forms, which is used by the plant as nutrient. Other bacteria can fix atmospheric nitrogen while free-living in the soil (examples Azospirillum and Azotobacter), thus enriching the nitrogen content of the soil. Fungi are also known to form symbiotic associations with plants (mycorrhiza). Many members of the genus Glomus form mycorrhiza. The fungal symbiont in these associations absorbs phosphorus from soil and passes it to the plant. Plants having such associations show other benefits also, such as resistance to root-borne pathogens, tolerance to salinity and drought, and an overall increase in plant growth and development. Cyanobacteria are autotrophic microbes widely distributed in aquatic and terrestrial environments many of which can fix atmospheric nitrogen, e.g. Anabaena, Nostoc, Oscillatoria, etc. In paddy fields, cyanobacteria serve as an important biofertiliser. Blue green algae also add organic matter to the soil and increase its fertility. Currently, in our country, a number of biofertilisers are available commercially in the market and farmers use these regularly in their fields to replenish soil nutrients and to reduce dependence on chemical fertilisers. HOTS 1 mark 1) Drinks like Whisky and Rum are more intoxicating than wine. Why? 2) Can we imagine a world without antibiotics? Explain briefly. 3) Bottled juices are clearer compared to homemade juice. Give reason. 4) Which one of these is a pertinacious infecting agent? a) Viroids, b) prions, c) protern, d) wat. 5) An organic farmer requires a bio control agent which is species specific and has no negative impact on non-target organism. Suggest the name of such a bio control agent. 6) Scientists have succeeded in introducing toxin gene of Bacillus thuringiensis into plants like cotton. What purpose is achieved through this action? 1 7)A small amount of curd was added to cold milk for converting it into curd. But milk did not get curdled. Why? 8) Consuming curd keeps the gastro-intestinal tract in tact. Give reason. 9) On fermentation the dough of idli and dosa puffs up. Which metabolic pathway is responsible for this process? 10) How do lactic acid bacteria help in increasing the nutritional quality of curd? 11) The excreta of cattle do not contain any cellulose but human excreta may contain cellulose. Why? 12) Antibiotics are always sold in combination with lactobacillus. Why? 13) Why are some microbes useful in detergent formulation? 14) Microbes release gases during metabolism. How will it help in the production of energy? 15) Why does 'Swiss-cheese' contain large holes?. 16)In what way the relationship between BOD and organic matter in Sewage will be useful in ecology? 17)Why do bottled fruit juices appear clearer than the home made ones? 18)What is the principle behind the conversion of milk into curd and partial digestion of milk protein performed by Lactic Acid Bacteria? 19)Wine and beer are different from whisky and brandy though they are all alcoholic beverages. What is the name of the process that brings out this difference? 20)Name the group of bacteria that are capable of living at high temperature above 1000C. How does it become possible by these bacteria?(1/2+1/2) 21)Why do doctors prescribe Streptokinases for brain hemorrhage patients? Mention the source of industrial production of this biomolecule.(1/2 + ½) 22)How are the holes produced in bread and cheese? 23.) Three water samples namely river water, untreated sewage water and secondary effluent discharge from a sewage treatment plan were subjected to BOD test. The samples were labeled A, B and C but the lab attendant did not note which BOD is for which water. The BOD values of three samples A, B and C were recorded as 20mg/L, 8mg/L and 400mg/L respectively. Which sample of the water is most polluted? Can you assign the correct label to each, assuming that the river water is relatively clean. 24. What are prions? 25. What is an antibiotics? 26. Identify the microbe grown on nutritive media in the given figure. 27. Why there are large holes in ‘Swiss cheese’? Name the bacterium used for this. 28. Give reasons why Staphylococci could not grow in unwashed culture plates of Alexander Fleming experiment? 29. Which bioactive molecule used as an immunosuppressive agent in organ-transplant patients? 30. What role do LAB play in our stomach? 31. What is toddy? 32. What are flocs? 33. Name the two plans initiated by Ministry of Environment and Forests to save major rivers of our country from pollution. 34. Which antibiotic was extensively used to treat American soldiers wounded in World War II ? 35. Name the free-living fungi that are very common in the root ecosystems which are effective biocontrol agents of several plant pathogens. 2 marks 1) A farmer was suggested to apply certain microbial culture in his field to increase the yield. Suggest the types of microbes he could use in his paddy field and how do they help in increasing the yield. 2) It was observed that certain plant roots are infected by fungus, in spite of this infection the plant showed increased growth and development. Give reason. 3) Arrange the steps in sewage treatment in proper sequence. a) Anaerobic sludge digestion b) 'Floc' formation c) Primary effluent in aeration tank d) Formation of biogas e) Constant agitation in presence of oxygen. f)Digestion by anaerobic bacteria 4) Bacteria that convert effluent into activated sludge play one more beneficial side.Mention the role 5) In the given figure label X & Y and explain the functioning of the below plant. 6) How does Monascus help in lowering blood-cholesterol? 7) Complete the given table Name of the organism 8. 9. Product/Enzyme/Bioactive molecule Aspergillus niger (i) (ii) Ethanol (iii) (iv) Cyclosporin-A Acetic acid Streptococcus (v) Monascus purpureus (vi) Name two alcoholic beverages produced in each of the following: (i) with distillation (ii) without distillation. Student tested the three water samples for BOD from different areas and he got following results. 47 46.5 46 mg/L 45.5 45 44.5 44 Sample A Answer the following based on above graph:- Sample B Sample C (i) Which water sample has more BOD? (ii) Which sample of the water is most polluted? 10. How Cyanobacteria increase fertility of soil? 11. What are the disadvantages of ‘conventional’ farming practices? How biocontrol helps in this regard? 12. State the role of following enzymes where they are used; (i) Lipases (ii) Streptokinase 13. Name the scientist who shared the nobel prize in 1945 for the discovery of antibiotic. From which fungus that antibiotic was extracted? 14.Fill in the blanks in the different columns of the table given below: Organic Acid Source Microbe a Lactobacillus Acetic acid b c Clostridium butylicum Citric acid d 15.What is the puffed up appearance of dosa due to? Which metabolic pathway involved in it? 16.What is the role of Baculoviruses in IPM? Name one of them used in this programme. 17.List the common uses of microbes in human welfare. 3 marks 1) For what significant properties the Baculoviruses are considered as best Biological control agent?. Mention its importance in organic farming. 2). Give one example and one use of the following:i) Free living fungi ii) Symbiotic fungi iii) Free living bacteria 3. How do biofertilizers enrich the fertility of the soil? 4. What is biological treatment? How it helps in treatment of effluents? 5. What are statins? Where are they produced from? How are they useful to man? 6. (i) Why is cow dung used in the generation of biogas? (ii) What are the major components of biogas? (iii) Name the common bacterium involve in biogas formation 5 Marks 1. Why is organic farming favoured these days? Describe the methods employed in the process. 2. Describe the treatment of sewage before it is discharged into a water body like a river. 3. (i) Why crystals of Bt not kill bacteria it self? How they kill insects? (ii) Do you think microbes can also be used as source of energy? If yes, how? 4. With the help of labeled diagram explain the production of biogas in your locality. Questions with hints of model answer Section - A: 1 Mark Questions [To be answered in one word or in one sentence] 1. Expand the following (each of 1 mark) i. STP Answer: Sewage Treatment Plant ii) BOD Answer: Biochemical Oxygen Demand. iii) IPM Answer: Integrated Pest Management iv. IARI Answer: Indian Agricultural Research Institute. v. LAB Answer: Lactic Acid Bacteria xiv) IVRI Answer: Indian Veterinary Research Institute 2. Why there is a big hole in Swiss cheese? Answer: The large holes in “Swiss cheese” are due to production of a large amount of CO 2 by a bacterium. ii) Ratna 3. Write the name of micro-organism responsible for production of citric Acid and Butyric Acid. Answer: i) Aspergillus niger (Fungus) – Citric Acid. ii) Clostridium butylicum (a bacterium) – butyric Acid 4. Name the fungus which is very commonly found in the root ecosystem used in treatment of plant diseases. Answer: Trichoderma. 5. Which fungus is effective bio control agents of several Plants Pathogens?. Answer: Trichoderma Section – B: 2 Marks Questions [To be answered in 20-30 words approximately] 1. Define “bioactive molecules”. Name two such molecules and give their sources. Answer: Bioactive molecules are those organic compounds which are produced on commercial scale by the microbes and are useful in the human welfare. Cyclosporin A - produced by fungus Statins - produced by yeast. 2. What is activated sludge? Answer: It is a measure for secondary treatment of sewage water. In this measure sludge is used which contain bacteria & algae. 7. Name a bioactive molecule used as an immunosuppressive agent in organ transplant patients also mention its source. Answer: Bioactive molecule – Cyclosporin A Source – Trichoderma Polysporum(Fungus) 10. What is mycorrhiza? How is it useful to some plants? Answer: Fungal association with the roots of some higher plants are known as mycorrhiza. It increase phosphorous absorption from the soil. Section – C: 3 Marks Questions [To be answered in 30-50 words approximately] 1. What are antibiotics? Give two examples. What is their significance? Answer: Antibiotics : - are the chemical substances produced by certain microbes, that can kill or retard the growth of other disease causing microbes. e.g – Penicillin, Streptomycin Significance: Antibiotics are used in the treatment of many human, animal and plant diseases. 2. Give the name of the microbes from which cyclosporine A and statins are obtained. cyclosporine A and status are useful to man? How Answer: Cyclosporin A is produced by the fungus Trichoderma polysporum Statins are produced by a yeast, Monascus purpureus. Uses – Cyclosporin A is used as an immuno suppressive agent during organ transplantation. Statins are used for lowering the levels of blood cholesterols, as it acts as a competitive inhibitor of the enzyme involved in cholesterol synthesis. 3. Name any three organic acids and the bacteria that produce them. Answer: Organic Acids i) Acetic Acid ii) Butyric acid iii) Lactic acid Bacteria Acetobacter aceti Clostridium butylicum Lactobacillus delbrueckii Section – D: 5 Marks Questions [To be answered in 80 -120 words approximately] 1. How do biofertilisers enrich the fertility of the soil? Answer: Biofertilizers are the organisms that enrich the nutrient quality of soil. Bacteria, cyanobacteria and fungi are the three groups of organisms used as biofertilisers. i) Bacteria a) symbiotic bacteria Rhizobium. b) Free living bacteria Azospirillum & Azotobactor. c) They fix the atmospheric nitrogen and enrich soil nutrients. ii) Cyanobacteria Cyanobacteria are autotrophic microbes widely distributed in aquatic and terrestrial environment. Many of them can fix atmospheric nitrogen and increase the organic matter of the soil through their photosynthetic activity. In paddy field cyanobacteria serves as important biofertilizer. Commercially fermers use these cyanobacteria in their fields to replenish soil nutrients and to reduce dependency on chemical fertilizers. Important examples are Anabaena, Nostoc and Oscillatoria. iii) Fungi/ Micorrhiza Fungi are also known to develop symbiotic relationship with plant roots. Such a relationship is called micorrhiza. Many members of the genus Glomus form micorrhizae. The fungus absorbs phosphorus from the soil and passes it to the plant. Plants having such association show other benefits alsoa) Resistance to root borne pathogens b) Tolerance to salinity. c) Tolerance to drought d) Overall increase in the plant growth and development. Ch.11 BIOTECHNOLOGY-PRINCIPLES & PROCESSES IMPORTANT CONCEPTS AND DEFINNITIONS1. Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans. 2. Biotechnology - The definition given by EFB is as follows: ‘The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services’. 3. Genetic engineering : Techniques to alter the chemistry of genetic material (DNA and RNA), to introduce these into host organisms and thus change the phenotype of the host organism. 4. Restriction enzymes: Enzymes that are used to cut DNA segment at a specific site are called restriction enzymes. 5. Exonucleases remove nucleotides from the ends of the DNA molecule. 6. Endonucleases make cuts at specific positions within the DNA molecule. 7. Plasmid - autonomously replicating circular extra-chromosomal DNA of any bacteria. 8. Origin of replication - a specific DNA sequence which is responsible for initiating replication is called origin of replication 9. Vectors - These are plasmid DNA or viruses act as vehicle to transfer the piece of DNA attached to it. 10. Palindromic Nucleotide Sequences - 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. 11. Gel electrophoresis - a technique which is used to separate the fragments of DNA is known as gel electrophoresis. 12. Transformation is a procedure through which a piece of DNA is introduced in a host bacterium 13. Insertional inactivation – The procedure of inserting a recombinant DNA within coding sequence of a functional gene which makes that gene inactive (unable to express) is called insertional inactivation. 14. Selectable marker- the gene encoding desirable information useful in identifying and eliminating nontransformants and selectively permitting the growth of the transformants is called selectable marker. 15. Micro-injection- a technique in which recombinant DNA is directly injected into the nucleus of an animal cell. 16. Biolistics or Gene gun - plant cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in a method known as biolistics or gene gun. 17. Bioreactors - bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial plant, animal or human cells. IDENTICAL TERMS1. Endonucleases / Exonucleases 2. Vectors / Plasmids 3. Genetic Engineering / Recombinant DNA Technology 4. Recombinant DNA / Recombinant Protein 5. Simple Stirred-tank Bioreactor / Sparged Stirred-tank Bioreactor IMPORTANT POINTS, PRINCIPLES AND THEORY1. Genetic engineering : Techniques to alter the chemistry of genetic material (DNA and RNA), to introduce these into host organisms and thus change the phenotype of the host organism. 2. Maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc. 3. Three basic steps in genetically modifying an organism — (i) identification of DNA with desirable genes; (ii) introduction of the identified DNA into the host; (iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny. TOOLS OF RECOMBINANT DNA TECHNOLOGY--1. DNA technology can be accomplished only if we have the key tools, i.e., restriction enzymes, polymerase enzymes, ligases, vectors and the host organism. 2. The first restriction endonuclease–Hind II 3. 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. 4. The convention for naming these enzymes is the first letter of the name comes from the genes and the second two letters come from the species of the prokaryotic cell from which they were isolated, e.g., EcoRI comes from Escherichia coli RY 13. 5. In EcoRI, the letter ‘R’ is derived from the name of strain. Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria. 6. Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA. 7. 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. For example, the following sequences reads the same on the two strands in 5' This is also true if read in the 3' ' direction. 5' —— GAATTC —— 3' 3' —— CTTAAG —— 5' 8. Restriction endonucleases are used in genetic engineering to form ‘recombinant’ molecules of DNA, which are composed of DNA from different sources/genomes. 9. When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of ‘stickyends’ and, these can be joined together (end-to-end) using DNA ligases Diagrammatic representation of recombinant DNA technology (A) Separation and isolation of DNA fragments : The cutting of DNA by restriction endonucleases results in the fragmentes 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. Hence, the smaller the fragment size, the farther it moves. 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 (you cannot see pure DNA fragments in the visible light and without staining). You can see bright orange coloured bands of DNA in a ethidium bromide stained gel exposed to UV light. 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. A typical agarose gel electrophoresis showing migration of undigested (lane 1) and digested set of DNA fragments (lane 2 to 4) (B) Cloning Vectors: Making many copies of rDNA is possible through multiplying the vector to which it has aligned. We are able to link an alien piece of DNA with bacteriophage or plasmid DNA, we can multiply its numbers equal to the copy number of the plasmid or bacteriophage. (C) The following are the features that are required to facilitate cloning into a vector. (i) Origin of replication (ori) : This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells. (ii) Selectable marker : In addition to ‘ori’, the vector requires a selectable marker, which helps in identifying and eliminating nontransformants and selectively permitting the growth of the transformants. Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for E. coli. The normal E. coli cells do not carry resistance against any of these antibiotics. (iii) Cloning sites: In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes. Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning. E. coli cloning vector pBR322 showing restriction sites (Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I, Cla I), ori and antibiotic resistance genes (ampR and tetR). Rop codes for the proteins involved in the replication of the plasmid. Alternative selectable markers have been developed which differentiate recombinants from nonrecombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, â-galactosidase (gene gets ‘inactivated due to insertion’ of alien DNA). This results into inactivation of the enzyme, which is referred to as insertional inactivation. The presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the â-galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies. (iv) Vectors for cloning genes in plants and animals: The tumor inducing (Ti) plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector which is no more pathogenic to the plants but is still able to use the mechanisms to deliver genes of our interest into a variety of plants. Similarly, retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells. So, once a gene or a DNA fragment has been ligated into a suitable vector it is transferred into a bacterial, plant or animal host (where it multiplies). (D) Competent Host (For Transformation with Recombinant DNA) 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 420C (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA. Other Methods:1. In a method known as micro-injection, recombinant DNA is directly injected into the nucleus of an animal cell. 2. 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 gene gun. 3. Another method uses ‘disarmed pathogen’ vectors, which when allowed to infect the cell, transfer the recombinant DNA into the host (E) Isolation and purification of the Genetic Material (DNA)Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus). Genes are located on long molecules of DNA interwined with proteins such as histones. The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease. Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. This can be seen as collection of fine threads in the suspension by spooling. (F) Amplification of Gene of Interest using PCRPCR stands for Polymerase Chain Reaction. In this reaction, multiple copies of the gene (or DNA) of interest is synthesised in vitro using two sets of primers (small chemically synthesised oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase. The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template. If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times, i.e., 1 billion copies are made. Such repeated amplification is achieved by the use of a thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus), which remain active during the high temperature induced denaturation of double stranded DNA. The amplified fragment if desired can now be used to ligate with a vector for further cloning. Polymerase chain reaction (PCR) : Each cycle has three steps: (i) Denaturation; (ii) Primer annealing; and (iii) Extension of primers (G) Obtaining the Foreign Gene ProductAfter having cloned the gene of interest and having optimised the conditions to induce the expression of the target protein, one has to consider producing it on a large scale. If any protein encoding gene is expressed in a heterologous host, is called a recombinant protein. The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory. The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques. Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100-1000 litres) of culture can be processed, was required. Bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial plant, animal or human cells. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen). (a) Simple stirred-tank bioreactor; (b) Sparged stirred-tank bioreactor through which sterile air bubbles are sparged A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. Alternatively air can be bubbled through the reactor. The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically. (H) Downstream ProcessingAfter completion of the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished product. The processes include separation and purification, which are collectively referred to as downstream processing. The product has to be formulated with suitable preservatives. Such formulation has to undergo 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. HOTS 1 marks 1 Restriction Enzymes are called “Molecular scissors”. In what context they are referred so? 2 In what way the following two plasmids differ? 1 3. What is the source of thermostable DNA polymerase and Name that polymerase. Why thermostable DNA polymerase is essential in PCR? 1 4 “The prophase I of meiosis plays a vital role in r-DNA formation” Justify the statement.1 5 Eukaryotes do not have restriction endonuclease, then how they manage with normal endonuclease enzyme?1 6What special feature do prokaryotes have to defend themselves from bacteriophages?1 7_____= Denaturation +______+Extenton.1 8.Complete the following palindrome sequence and name the restriction endonuclese that recognises this. 5’ G ? A ? T C 3’ 3’ C ? T ? A G 5’ 9. Which is not a tool of Recombinant DNA technology. a) Restriction enzyme b) Vector c)Bioreactor 10. In what stage of meiosis recombination of DNA takes place? 11. How has EFB defined biotechnology? 12. Which group of enzymes known as molecular scissors? 13. From which organism restriction endonuclease was first extracted? 14. How is a DNA fragment amplified? 15. Name the common substance used as matrix for gel electrophoresis. 16. Which of the following DNA sequences would a Restriction enzyme recognize and cut? (a) ATGCAC (b) GATATC (c) TAGATA (d) AATATA TACGTG CTATAG ATCTAT TTATAT 17. Who isolated the antibiotic resistance genes from bacteria? 18. What are recognition sequences? 19. What is elution in the process of separation of DNA fragments? 20. Name the chemical used to stain DNA in gel to view them. 21. What is Rop in cloning vector pBR322? 22. Which enzyme is used to treat the fungal cells during isolation of DNA? 23. Identify the type of bioreactor given in below figure:- 24. Why does DNA move towards 25. What is gene gun? the anode in gel electrophoresis? 2marks 1. Is advisable to use different restriction endonucleases to cut the vector DNA and source DNA? Why? 2. What will happen if more than one recognition sites are present in a vector? 3.Uncontrolled recombinant DNA technology experiments is dangerous to mankind. Comment on it. 4. + = -------------------------------- Foreign DNA + plasmid = …………??………… Complete the above sequence of diagrammatic representation and name it. 5. (a) Which is the most commonly used matrix in gel electrophoresis ? (b) What is the source of it? 6 Find the ‘odd one out and write why that is ‘odd’ (a) Sal I, Pst I, Cla I, BamH I, pBR 322 (b) Bacteria, Virus, Gene-gun, Fungi 7 Detect the mismatch from the following and replace the wrong match with a right one (a) ECOR I – Gel electrophoresis (b) Ethidium Bromidqe Bacteria (c) Lysozyme Restriction enzyme (d) Palindrome sequence Fungi 8.Write the use of the following in Biotechnology. (a) Chilled ethanol (b) Microinjection (c) Bioreactor (d) plasmid 9. Is there any difference between recombinant DNA and recombinant protein? Support your answer. 10. What are selectable markers? Give two examples. 11. What are the optimal conditions bioreactor provides for the achieving the desired product of gene? 12. Few gaps have been left in the following table showing certain terms and their meanings. Sl.No Terms Meanings 1. ---------------any protein encoding gene is expressed in a heterologous host 2. lysozyme ------------------------------------------------3. ---------------In this recombinant DNA is directly injected into the nucleus of an animal cell. 4. Ti ---------------------------13. How you overcome inclusion and multiplication of undesirable genes along with the desired genes by hybridisation procedures used in plants? What techniques are included in this process? 14. A scientis cultured the recombinant DNA bearing gene for resistance to an antibiotic ampicillin is transferred into bacterial cells on agar plates containing ampicillin.Which one will grow on this medium? What you call such type genes in bacterial cells? 15. How you Isolate the Genetic Material (DNA) from plant cells? 16. Why the bacterial cells must first be made ‘competent’ to take up DNA? List the methods used to achieve this Process. 17. How has Agrobacterium tumifaciens been suitably modified to act as a cloning vector? 18. Name the 1,2,3 And Enzyme 4 in given below diagram. 19. What are three basic steps in genetically modifying an organism? 3 MARKS 1. Give the correct term for the following; (a) Replacing a defective mutant allele with a normal functional allele /gene. (b) Increasing the copies (content) of a gene (c) Preventing m-RNA translation (d) Specific pattern of base pairs that are recognized by restriction enzymes. (e) Mobile genetic element (f) One DNA copies itself and produces two 2. Name the enzyme involved in the following process: (a) Repeated amplification of DNA fragments. (b) Formation of short piece of RNA strand for annealing. (c) Breaking of bacterial cell to release DNA and other macromolecules. (d) Cutting and rejoining DNA fragments. (e) Formation of m-RNA. (f) Joining of foreign DNA fragments with plasmid. 3. Explain the following, emphasizing their formation. (a) rDNA (b) cDNA (c) dsRNA 4. Name the diagram and label the parts ‘a’ to ‘e’. 5. (i) Label the following diagram & Identify the selectable markers (2) (ii) What is the role of polymerase? (1) 6. Two of the steps involved in producing rDNA are mentioned below. Write the missing steps in its proper sequence? a. Isolation of DNA, b.---------------------------------c.---------------------------------d.--------------------------------e. ligation of the DNA fragment into a vector, f.--------------------------------------------------g.-------------------------------------------------. 7. Describe the naming of restriction enzymes with an example. 8. What is insertional inactivation? Which enzyme is used in this process? What type of colour it gives in its reaction? 9. What Polymerase Chain Reaction? Name the steps involved in this process? Name the bacteria used to isolate thermostable polymerase enzyme. 5 MARKS 1. (a) Explain how recombinants and non- recombinants are differentiated on the basis of colour production in the presence of a chremogenic substrate. Name that procedure. (b) Describe the temperature treatment (-3 step-) that enhances the bacteria to take up the rDNA. 2. What features are required to facilitate as cloning vector? Describe in brief. 3. Diagrammatically represent the experimental set up in cloning and expressing a human genome like growth hormone in to a bacterium E.coli Questions with hints of model answers Section - A: 1 Mark Questions [To be answered in one word in one sentence] 1. What is plasmid? Answer: Plasmid is autonomously replicating circular extra chromosomal DNA. 2. Write any two ways of introducing foreign DNA into host cell. Answer: i) Micro-injection ii) biolistics/ gene gun. 3. Which compound is used for staining DNA in gel? Answer: Ethidium Bromide. 4. Name the enzyme that digest bacterial cell wall in genetic engineering. Answer: Lysozyme. Section – B: 2 Marks Questions [To be answered in 20 to 30 words approximately] 1. A farmer found that agrochemicals cause pollution of soil and water and are too expensive. What alternative should be use? Answer: Farmer should use genetically modified crops and sould have organic farming as a possible solution for given problems. 2. What are the key tools needed for the recombinant technology? Answer: The key tools needed for the recombinant technology to be accomplished are:i) cell culture with desired DNA ii) restriction enzymes iii) DNA polymerase iv) Ligases v) Vector vi) Host organism/ Cell 3. To which class of enzyme restriction enzyme belong and write its types. Answer: Restriction enzyme belong to a class of enzyme, called nuclease. There are following two kinds of restriction enzymes:a) Exonucleases, which remove nucleotides from two ends of DNA. b) Endonucleases, which cut the DNA at specific position anywhere in its length. 8. What is palindrome nucleotide sequence? Answer: The recognition sequence is a palindrome, where the sequence of base pairs reads the same on both the DNA strands. Example5’- GAATTC- 3’ 3’- CTTAAG- 5’ 4. What is vector in genetic engineering? Write its one property. Answer: Plasmid and bacteriophages are the commonly used vectors, the vector should have a few (or) at least one unique recognition site, to link the foreign/ alien DNA. 5. What is marker? Answer: A marker is a gene, which helps in selecting those host cells which contain the vector (transformant) and eliminating the non-transformants. Section – C: 3 Marks Questions [To be answered in 30 to 50 words approximately] 1. Why are cloning vectors necessary in cloning? Name any two such vectors that are used in experiments with Escherichia coli. Answer: Cloning vectors are necessary in cloning because these are the vehicle for cloning. The vector carries a foreign DNA sequence into a given host cell. Bacterial plasmids and bacteriophages are considerd the most useful. Name of two vectors are:i) PBR 322 and ii) PUC (named after University of California) is reconstructed plasmid vector. 2. What is genetic engineering? List the steps involved in rDNA technology? Answer: Genetic engineering is a technique of molecular biology which includes deliberate manipulation of genetic make up of an organism, synthesis of new DNA, repair of DNA etc. Recombinant DNA (rDNA) technology involves the following steps:i) Isolation of DNA. ii) Fragmentation of DNA by restriction endonuclease iii) Isolation of the desired DNA fragment. iv) Amplification of the gene of interest. v) Ligation of two DNA fragment into a vector using DNA ligase. 4. Which convention do you use to write name of restriction enzymes? Answer: first of all scientific name, strain and then order of enzyme isolation is written in roman. Example- ECoRI where, E- Escherichia (Genus) Co- coli (species) R- Strain of organism I- order of enzyme isolation. 5. What is palindrome in DNA? Give one example. Answer: Palindrome in DNA is a sequence of base pairs that pairs that reads same in two strands when orientation of reading is kept the same. 5’- GAATTC- 3’ 3’- CTTAAG- 5’ These two strands read same in 5’-3’ direction as well as in 3’-5’ direction. 6. Define the terms: i) Recombinant DNA iii) Plasmid v) Transgenic ii) sticky ends iv) gene gun vi) Bioconversion Answer: i) Recombinant DNA: It is the DNA formed by combining DNA from two different organisms. ii) Sticky ends: refers to the single stranded proteins at the ends of DNA when cut by a restriction enzyme. iii) Plasmid: extra chromosomal fragments of DNA present in bacteria which is circular. iv) Gene Gun: bombarding micro projectiles coated with foreign DNA with great velocity into the target cell. v) Transgenic: genetically modified organisms that contain a foreign gene. vi) Bio conversion: raw materials are biologically converted into specific products using microbial, plant or animal cells. Section – D: 5 Marks Questions [To be answered in 80 to 120 words approximately] 1. What are Bioreactors? Write its types and describe them. Answer: Bioreactors: These bio appliances could be considered as vessels in which certain raw materials are biologically converted into specific products by microbes (or) plants/ animals cells and/or their enzymes. Two common types of bioreactors are the following:a) Simple stirred tank bioreactor b) Sparged stirred tank bioreactor Stirred tank bioreactors are the most commonly used bioreactors. In a simple stirred tank bioreactor, there is usually a cylindrical vessel (or) a vessel with a curved base to facilitate mixing of the content. But in the sparged stirred tank bioreactor sterile air bubble are sparged. 2. What is rDNA? Explain the technique of cloning DNA. Answer: To alter the chemistry of genetic material by gene cloning and gene transfer. Techniques involved in Recombinant DNA:i) Isolation of DNA. ii) Fragments of DNA by restriction Endonucleases. iii) Isolation of desired DNA fragments. iv) Amplification of the gene. v) Ligation of the DNA fragments. vi) Transfer of recombinant DNA into the host. vii) Culturing the host cells. viii) Extraction of desired product.