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12. Biotechnology
Biotechnology the product of interaction between the biological science and technology. It is infact, an applied branch of biology. The term biotechnology was first used by Karl Ereky in 1919 to describe a process for large scale production of pigs. Definition - The development and utilization of biological forms, products or processes for obtaining maximum benifits to man and other forms of lifeā. According to OECD (Organization for Economic Cooperation and Development, 1981)- āIt is the application of scientific and engineering principles to the processing of materials by biological agents to provide goods and service to the human welfare. Development of biotechnology in terms of its growth, occurred in two phases viz, Traditional biotechnology and Modern biotechnology. Traditional biotechnology (old biotechnology) : It was primarily based on fermentation technology using microorganisms as in the preparation of curd, ghee, soma. Modern biotechnology (new biotechnology) : The combination of biology and production technology based on genetic engineering.
Principles and Processes of Biotechnology :
Modern biotechnology is based on two core techniques viz. genetic engineering and chemical engineering. o Chemical engineering It deals with maintaining sterile environment for manufacturing variety of useful products including vaccines, antibodies, enzymes, organic acids, vitamins, therapeutics, etc. o Genetic engineering It is defined as the manupulation of genetic material towards a desired end and in a directed and predetermined way, using in vitro process. Therefore, the genetic engineering is alternatively called recombinant DNA technology or gene cloning. Technique of gene cloning and rDNA technology : In gene cloning, a gene of known function can be transferred from its normal location into a cell (that of course does not contain it) via a suitable vector. The transferred gene is replicated normally and is handed over to the next progeny. A. Tools and techniques for gene cloning/ rDNA technology : The basic requirements for the technique are as follows: I. Different instruments (devices) :
- Macromolecule such as DNA, RNA, proteins, etc. are synthesized in the living cells which vary in their molecular weight
- The techniques used on the basis of molecular weight, are gel permeation, osmotic pressure, ion exchange chromatography, spectroscopy, mass spectrometry, electrophoresis, etc
- Electrophoresis is the separation of charged molecules, applying an electric field. It is applied for the separation of DNA, RNA and proteins. DNA being negatively charged, migrates to anode. Small fragments of DNA molecules, move faster and thus separate faster. Use of Agarose gel electrophoresis, PAGE, SDA PAGE are the different methods.
Polymerase chain reaction (PCR) : āŖ Polymerase chain reaction (PCR) is another device used for gene cloning or gene multiplication in vitro. It is the amplification of gene of interest, through PCR. āŖ In 1985, Kary Mullis made an important discovery (contribution) in the form of an extremely powerful technique called polymerase chain reaction (PCR). PCR can generate a billion copies of the desired segment of DNA or RNA. āŖ PCR is in vitro amplification of a desired DNA segment, which requires : DNA containing the desired segment to be amplified, several molecules of four deoxyribonuclueoside triphosphates (dNTPs), excess of two primer molecules, heat stable DNA polymerase and appropriate quantities of Mg++ ions. Mechanism of PCR: At the start of PCR, the DNA segment, and excess of two primer molecules, four deoxyribonucleosides triphosphates and the thermostable DNA polymerase are mixed together in āeppendorf tubeā Step i : The reaction mixture is heated to a temperature (90ā98 oC) to separate two strands of desired DNA. This is called denaturation. Step ii : The mixture is allowed to cool (40ā60 oC) that permits pairing of the primer to the complementary sequences in DNA. This step is called annealing. Step iii : The temperature (70ā75 oC) allows thermostable Taq DNA polymerase to use single-stranded DNA as template and adds nucleotides. This is called primer extension. It takes arround two minutes duration Denaturation (90- 98 Ā°C) 1 copy of desired gene DNA strands separated Annealling of primers (40Ā°- 60 Ā°C) Replicating DNA strands using Tag polymerase (70Ā°- 75 Ā°C) 2 copies of desired gene (renaturation) Second cycle continues to form 4 copies of desired DNA Cycle continous to produce 2n copies of DNA
When one reads the sequence in opposite direction (3 ā to 5 ā or 5 ā to 3 ā ) it is identical/ same. DNA showing palandromic sequence A sequence with this type of symmetry is called a palindrome. When the enzyme EcoRI attacks this palindrome, it breaks each strand at the same site in the sequence, which is indicated by the arrow between the A and G residues. 3 ā ---- C T T A A G 5 ā 5 ā ---- G A A T T C 3 ā Restriction enzymes either cut straight across the DNA in the region of palindrome to give blunt ends or cuts producing short, single stranded projections at each end of DNA to produce, cohesive or sticky ends or staggered ends. B. Cloning vectors (vehicle DNA) - Vectors are DNA molecules that carry a foreign DNA segment and replicate inside the host cell. Vectors may be plasmids, bacteriophages (M13, lambda virus), cosmid, phagemids, BAC (bacterial artificial chromosome), YAC (yeast artificial chromosome), transposons, baculoviruses and mammalian artificial chromosomes (MACs),. Most commonly used vectors are plasmid vectors (pBR 322, pUC, Ti plasmid) and bacteriophages (lamda phase, M13 phage). i. Plasmid : The plasmids most commonly used in recombinant DNA technology are those that replicate in E. coli. ii. Plasmid vectors for plants : An important vector for carrying new DNA into many types of plants is a plasmid that is found in Agrobacterium tumefaciens. This bacterium lives in the soil and causes a plant disease called crown gall, which is characterized by the presence of over- growths, or tumors, in the plant. A. tumefaciens contains a plasmid called Ti (for tumor-inducing). The Ti plasmid contains a transposon, called T DNA, which inserts copies of itself into the chromosomes of infected plant cells. C. Competent host : (cloning organism) used are usually the bacteria like Bacillus haemophilus , Helicobacter pyroli and E. coli. Mostly E. coli is used for the transformation with recombinant DNA
Methodology for rDNA technology
The steps involved in gene cloning are as follows : a. Isolation of DNA (gene) from the donor organism : i. The desire gene to be cloned has to be obtained from the source organism (donor). Initially the cells of the donor organism are sheared with the blender and treated with suitable detergent. Genetic material from the donor is removed isolated and purified by using several techniques. ii. Isolated purified DNA is then cleaved by using restriction enzymes particularly Restriction Endonucleases (RE). These enzymes cleave DNA at specific sites, called restriction sites and break the DNA into fragments.
b. Insertion of desired foreign gene into a cloning vector (vehicle DNA) : The foreign DNA or passanger DNA is now inserted into a cloning vector or vehicle DNA. The most commonly used cloning vectors are plasmids of bacteria and the bacteriophage viruses like lamda phage and M13. The most commonly used plasmid is pBR 322. Plasmids are isolated from the vector. The combination of vector DNA and foreign DNA is now called Recombinant DNA or Chimeric DNA and the technology is reffered to as rDNA technology c. Transfer of rDNA into suitable competent host or cloning organism : Finally the recombinant DNA is now introduced i.e. transferred for expression into a competent host cell of the suitable cloning organism which is usually a bacterium. Host cell takes up naked rDNA by process of ātransformationā d. Selection of the transformed host cell : The transformation process generates a mixed population of transformed (recombinant) and non- transformed (non-recombinant) host cells. For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector is employed. For example, PBR322 plasmid vector contains different marker gene. e. Multiplication of transformed host cell: Once transformed, host cells are separated by the screening process. In this step the transformed host cells are introduced into fresh culture media. f. Expression of the gene to obtain the desired product: The next step involves the production of desired products like alcohol, enzymes, antibiotics, etc. Finally the desired product is separated and purified through downstream processing using suitable bioreactor
Applications of Biotechnology:
Biotechnology is an umbrella term covering a broad spectrum of scientific applications used in many sectors, such as health and agriculture. Industry, environment and genomics
- Hakura et al, chemically synthesized DNA sequence of insulin for two chains A and B and separately inserted into two PBR322 plasmid vector. Vaccine production:
- A vaccine is a biological preparation that provides active acquired immunity against a certain disease.
- vaccine consists of a biological agent that represents the disease- causing microorganism. It is often made from a weakened or killed form of the microorganism
- Vaccines have eliminated small pox, polio and other deadly diseases for the last several decades. Oral vaccines: a novel approach
- An exciting invention is production of āmelt in the mouthā vaccines that can be administered by placing them under your tongue that delivers it into the blood stream.
- The most important example is the production of flu vaccine by Bacillus which melts in the mouth.
- The tremendous benefit of such vaccines, is the comfort of administeration, low cost and ease of storage. b. Agriculture :
- Application of Biotechnology in Agriculture involves scientific techniques such as Genetically Modified Organisms, Bt Cotton, Pest Resistant Plants.
- Tissue Culture is used in Micropropagation i.e. large-scale propagation of plants in very short durations. Tissue culture technique is also the best method for storing germplasm and maintaining a specific genetic type (Clone).
- This technique is used in those plants, which produce recalcitrant seeds or produce highly. c. Gene therapy is the treatment of disease by replacing, altering or supplementing a gene that is absent or abnormal and whose absence or abnormality is responsible for the disease.
- Replace missing or defective genes;
- ā¢ Deliver genes that speed the destruction of cancer cells;
- ā¢ Supply genes that cause cancer cells to
There are more than 5000 different human genetic diseases known to be caused by single gene defects e.g. sickle cell anaemia, thalassemia, Tay-sachās disease, cystic fibrosis, Huntingtonās chorea, haemophilia, alkaptonuria, albinism, etc. Gene therapy is being used in many ways. For example, ā¢ Supply genes that cause cancer cells to revert back to normal cells; ā¢ Deliver bacterial or viral genes as a form of vaccination; ā¢ Deliver DNA to antigen expression and generation of immune response; ā¢ Supply of gene for impairing viral replication; ā¢ Provide genes that promote or impede the growth of new tissue; and Forms of gene therapy a. Germ line gene therapy : In this method healthy genes can be introduced into germ cells like sperms, eggs, early embryos. It allows transmission of the modified genetic information to the next generation
b. Somatic cell gene therapy : In this type the gene is introduced only in somatic cells like bone marrow cells, hepatic cells, fibroblasts endothelium and pulmonary epithelial cells d. Genetically Modified Organisms (GMOs) : I. Transgenic Plants o Traditional breeding programmes involve sexual crosses, which resulted in the high quality of present day food plants such as wheat, rice, corn, potato, etc. o More recently, biotechnological approaches have been applied to these plants to create genetic variations that are beneficial for mankind o First transgenic plant produced was tobacco. More then 60 transgenic dicot plants and several monocot plant like maize, oat, rice, wheat are known. Tomato, soybean, potato, sugar beet, grapes, brinjal, cotton are other transgenic plants. a. Insect pest resistance : It can help farmers to reduce their use of chemical pesticides, which in turn can reduce the cost of producing food. an alternative has been available for more than 30 years which is a biological insecticide from the bacterium, Bacillus thuringiensis (Bt). Bt cotton is one of the best transgenic plants known for its insect resistance property. Insect resistant plants contain either a gene from B. thuringiensis or the cowpea trypsin inhibitor gene. The gene called cry gene present in B. thuringiensis produces a protein that forms crystalline inclusions in bacterial spores. When ingested by a susceptible insect, a combination of high pH and the enzyme proteinase of the insectās midgut, processes them hydrolytically to release the core toxic fragments. Bt toxin activity has been against many species of insects within the orders of Lepidoptera, Diptera, and Coleoptera. the gene of Ī±-amylase inhibitor (Ī±Al-Pv) has been isolated from adzuki bean ( Phaseolus vulgaris ) and transferred to tobacco and this gene works against pests like Zabrotes subfasciatus and Callosobruchus chinensis. b. Improved nutritional qualities (biofortification) : Transgenic plants have also been produced to provide functional food and neutraceuticals blindness that results from a lack of vitamin A. This vitamin is abundant in milk and in vegetables such as carrots. Swiss researchers created transgenic rice (golden rice) and transgenic mustard (golden mustard) varieties that are high in vitamin A. The golden colour is due to vitamin A. Improvement in oil content and oil quality of oil crops like soybean, oil palm, rapeseed and sunflower, have been achieved by transfer of ā Arabidopsis genes. For production of transgenic crops that will produce food rich in iron, an iron storage protein (ferritin) is targeted. Ferrritin is found in many animals, plants and bacteria. The genes for ferritin protein isolated from soybean and Phaseolus have been transferred to rice. c. Modification in Post-harvest characteristics In the tomato the enzyme polygalacturonase breaks down the cell wall constituent- pectin, leading to softening of fruit during ripening. Thus, the fruits are easily bruised and damaged on shipment. By inhibiting the polygalacturonase by antisense genes, the tomato (genetically modified tomatoes are called Flavr savr tomatoes) can remain fresh d. Plants as factories : To produce novel biochemicals and vaccines (Biopharmaceuticals) , plants are potential factories or bioreactors for high value biochemicals like starch, sugar, lipids, proteins.
- Wool quality and quantity 6. Disease resistance in animals 7. Production of low-cost pharmaceuticals c. Transgenic cattle for food production Researchers introduced additional copies of bovine beta or kappa casein into dairy cattle and evaluated the effect on milk production and composition. Transgenic offspring had an 8 to 20% increase in beta casein and a two-fold increase in kappa casein d. Transgenic cattle for human therapeutic production : A second application for genetically modified cattle is the production of human therapeutic proteins. The mammary gland in dairy cows is an excellent protein production factory. On the other hand, one transgenic cow would be more than sufficient for production of annual world supply of factor IX (plasma thromboplastin component) that is used in the treatment of haemophilia. In 1990 Tracy , the transgenic cow was born in Scotland, and could produce a human protein in her milk for human therapeutic e. Transgenic Sheep : Gene transfer technology is applied to sheep to produce transgenic sheep which are able to achieve better growth and meat production as well as to serve as bioreactors Bacterial genes, cys E and cys M , are concerned with biosynthesis of cysteine amino acids involved in formation of keratin protein found in wool. Both these genes are identified, cloned and introduced in sheep to increase wool production and to improve the quality of wool f. Transgenic pigs : The objective of gene transfer in pigs is to increase growth and meat production and to act as bioreactors Pigs are regarded as the most suitable animals to be bred for heart transplant because a pigās heart is about the same size as a human heart, and pig heart valves have been used in human heart surgery for over a decade g. Transgenic fish : The commercially important fish like Atlantic salmon, catfish, goldfish, Tilapia , zebra-fish, common carp, rainbow trout, etc. are transfected with growth hormone, chicken crystalline protein and E.coli hygromycin resistance gene. Transgenic fish showed increased cold tolerance and improved growth h. Transgenic chicken : They could be used to improve the genetic make-up of existing strains with respect to built-in ( in vivo ) resistance to viral and coccidial diseases, better feed efficiency, lower fat and cholesterol levels and high protein containing eggs, and better meat quality. Bioethics :
- Ethics usually deals with the matters related to socially acceptable moral duty, conduct and judgement. In otherwords, it helps to regulate the behaviour of cummunity by some set of standards
- Bioethics helps to study moral vision, decision and policies of human behaviour in relation to biological phenomena or events.
- Ethics deals with āLifeā e.g. in vitro fertilization, sperm bank, gene theropy, cloning, gene manipulations, euthanasia, death, maintaining those who are in comatose state, prenatal genetic selection
- Use of animals causes great sufferings to them; voilation of integration of species caused due to transgenosis; indiscriminate use of biotechnology pose risk to the environment, health and biodiversity.
- These include the effects on non-target organisms, insect resistance crops, gene flow and the loss of diversity. Ethics in biotechnology also includes the general subject of what should and should not be done in using recombinant DNA technique.
Effects of Biotechnology on the Environment :
a. Herbicide Use and Resistance : Effects on the environment are a particular concern with regard to GMO crops and food production. One such herbicide that has already been added is RoundUp. Crops of RoundUp- ready soybeans have already been implemented into agricultural practices, possibly conferring RoundUp resistance to neighboring plants. b. Effects on Untargeted Species : Bt corn, which produces its own pesticide, is also in use today. It has adverse effects on Monarch butterfly populations, which are not the original target of the pesticide. It can also have unintentional effects on neutral or even beneficial species.
Effects of Biotechnology on Human Health
a.Allergies : Researchers used a gene from the Brazil nut to increase the production of Methionine in soya beans. The insertion of this gene inadvertently caused allergic reactions to the soya bean in those with known nut allergies b. Long-Term Effects : Because GMO technology has been available for such a short amount of time, c. New Proteins : Proteins that have never been ingested before by humans are now part of the foods that people consume every day. This shows that the vast advances in life sciences and our multicultural and pluralistic modern societies create numerous bioethical problems requiring some stringent regulation. In terms of GMOs, the Indian Government has set up the Genetic Engineering Approval Committee (GEAC). This organization makes decisions regarding the validity of research involving GMOs and addresses the safety of GMOs introduced for public use.
Biopatent and Biopiracy :
a. Biopatent : ā¢ Patent is a special right granted to the inventor by the goverment. Patent is a personal property of inventor. It can be sold like any other property. A patent consists of three parts - grant (aggrement with the inventor), specification (subject matter of invention) and claims (scope of invention to be protected). ā¢ Biopatent is a biological patent. Biopatents are awarded for strains of microorganisms, cell lines, genetically modified strains, DNA ā¢ DNA sequences, biotechnological processes, product processes, product and product applications. ā¢ Biopatents are awarded to recognize real innovative contributions made by the inventor to the cause of human welfare. ā¢ Indian patent allows āprocess patentā and not the āproduct patentā. Biopatent allows the patent holder to exclude others from making, using, selling or importing protected invention for a limited period of time. ā¢ Duration of biopatents is five years from the date of the grant or seven years from the date of filing the patent application, which ever is less.
Multiple Choice Questions
- The term molecular scissors to a) Recombinant DNA b) Restriction enzymes c) Taq polymerase d) Palindrome nucleotide sequences
- The sticky ends of a fragmented DNA molecule are made up of a) Calcium salts b) Endonuclease enzyme c) Unpaired bases d) Methyl groups
- The term chimeric DNA refers to a) DNA with overhanging stretches b) DNA with palinadromic sequence c) a recombinant DNA d) Molecular scissors
- Gel electrophoresis is used for a) Construction of recombinant DNA by joining with cloning vectors b) Isolation of DNA molecules c) Cutting of DNA into fragments d) Separation of DNA fragments according to their size
- In plasmid PBR 322 BR stands for a) Baculovirus and Retrovirus b) Boyer and Reed c) Boliver and Rodrigu d) Bacillus and Rhizobium
- ā¦ā¦ā¦ā¦. a crown gall bacterium is called as ānatural genetic engineerā of plants. a) Esherichia coli b) Streptomyces albus c) Agrobacterium tumefaciens d) Azotobacter
- The term ācompetentā refers to a) increasing the competiting between cells b) making cell impermeable for DNA c) increasing the efficiency with which DNA enters the bacterium through pores in its cell wall d) making cell permeable for divalent cations
- Enzyme Taq polymerase used in PCR has been isolated from bacterium a) Agrobacterium tumefaciens b) Thermus aquaticus c) Streptomyces albus d) Escherichia coli
- Which peptide hormone produced by ļ¢- cells of istets of Langerhans of pancreas? a) Insulin b) Interferons c) Erythropoietin d) Relaxin
- In a polymerase chain reaction, temperature require for the steps i) Denataration ii) Annealing and iii) Extension are respectively a) i) 94Ā°- 98 ā ii) 40Ā°- 60 ā iii) 70Ā°- 75 ā b) i) 40Ā°- 60 ā ii) 70Ā°- 75 ā iii) 94 Ā°- 98 ā c) i) 94 Ā°- 98 ā ii) 70Ā°- 75 ā iii) 40Ā°- 60 ā d) i) 70Ā°- 75 ā ii) 94 Ā°- 98 ā iii) 40Ā°- 60 ā
- Golden rice is yellow in colour due to presence of a) riboflavins b) ļ¢ ā carotene c) vitamin B 12 d) complex genetic material
- A transgenic food crop which may help in solving the problem of high blindness in developing countries is a) Bt cotton b) golden rice c) Flavr save d) Bt corn
- What causes the inactive form of Bt toxin i.e. Protoxin to get converted into its active form in the body of an insect? a) Temperature of the gut b) Enzymes present in the saliva c) Alkaline pH of the gut d) Acidic pH of the gut
- First genetically modified plant commercially released in India is a) golden rice b) flavr savr c) Bt brinjal d) Bt cotton
- Which of the following genes were introduced in cotton to product it from cotton bollwarms? a) Cry Ac and Cry Ab b) Bt Ac and Bt Ab c) Cry I Ac and Cry II Ab d) Nif genes
1 mark questions
1) Define polymerase chain reaction.
2) How will you distinguish between Exonuclease and Endonuclease?
3) Give two examples of Biopiracy.
4) Define Bioethics?
5) What is gene therapy?
6) Enlist the biological tools of recombinant DNA technology.
7) Write down the vectors involved in r-DNA technology.
8) Name the enzyme used in extension step of PCR technique.
9) Which cells of the body produce insulin?
10) Name any two diseases in which somatic cell gene therapy is used.
11) Which is most widely used vector for producing Bt crops?
12) What is electrophoresis?
13) Write down any two features of plasmid being used as a cloning vector.
14) Name two resistant plants produced by using r-DNA technology.
15) which enzyme is called as molecular scissor?
16) Define genetic engineering.
17) What is the full form of EcoRI?
18) Can you recall meiosis and indicate at what stage a recombinant DNA is made?
19) Give any one example of competent host.
20) Name the vector which is used in production of human insulin through r-DNA technology.
2 mark questions
1) Write short note on Biopatent.
2) State how blunt ends and sticky ends are produced.
3) Give the basic requirements of PCR techniques.
4) What are Palindromes? Give one example Palindromes.
5) Enlist the applications of biotechnology.
6) Write short note on Biopiracy.
7) What are competent host? What does the word ācompetentā refer to?
8) Give the name and role of transgenic food crop which help in solving the problem of night
blindness.
9) Name the first transgenic cow and the human protein found in the milk of that cow.
10) Explain the types of gene therapy.
11) What is vaccine? Give role of vaccine.
12) What is full form of GEAC? Give its two objectives.
13) Define nuclease. Give two types of nucleases.
14) Write down the most commonly used cloning vectors? Define transformed cell.
15) What is name of iron storage protein? Give the two names of the plants from which they are
isolated.
16) Write short note on Bioethics.
17) Write short note on āFlavr savr tomatoā.
18) Expand YAC and MACs in Biotechnology.
19) Give the roles of the following in Biotechnology.
a) Restriction Endonuclease.
b) Gel-electrophoresis.
3 mark questions
1) Describe the steps involved in PCR technique.
2) What are the main objectives of transgenic animals for improved animal breeding
programmes?
3) Describe the applications of animal Biotechnology.
4) Mention the important properties which a good vector must possess.
5) Explain the process of formation of transgenic plants.
6) Give the example and name of bacterium melt in the mouth vaccine. Write two benefits of
oral/ edible vaccine.
7) What are cry genes? In which organism are they present. Give the role of cry gene.
4 mark questions
1) With the help of diagram explain the steps involved in r-DNA technology.
2) Define gene therapy. Give its applications.
3) Enlist and write in brief about the different biological tools required in r-DNA technology.
4) Describe in brief advantages of GM food crops.
5) Explain the use of transgenic animals indicating the applications of Biotechnology.
6) Describe the effects of biotechnology on human health.
7) Give reason-
i) How are the transgenic mice used in cancer research?
ii) Why pigs are regarded as suitable animals to be bred for heart-transplant?
iii) How farm animals are benefited to mankind?
iv) What type of changes in the gut of insects on consuming cry protein?
