BIOTECHNOLOGY: PRINCIPLES, PROCESSES AND APPLICATIONS

1. BIOTECHNOLOGY:PRINCIPLES, PROCESSES AND APPLICATIONS PROJECT DESIGNERS G R KRISHNA CHAND AVATAR ROLL - 8 GINNGAIHLIAN ROLL - 7

2. CERTIFICATE This is to certify that this project entitled “BIOTECHNOLOGY DEFINITION, PRINCIPLES AND PROCESSES” has been designed by both of us : Ginngaihlian R. No- G R Krishna Chand Avatar R. No.- to the best of our abilities for the partial fulfillment of the requirements of AISSC Examination 2012-13. It is a comprehensive work carried out under the supervision of Ms Lois Varghese and her satisfaction. DATED : J C LALSANGZUALA PLACE : PRINCIPAL JNV, Thenzawl

3. CERTIFICATE This is to certify that this project entitled “BIOTECHNOLOGY DEFINITION, PRINCIPLES AND PROCESSES” assigned to Ginngaihlian R. No- G R Krishna Chand Avatar R. No.- of Class XII (Science) for the partial fulfillment of the requirements of AISSC Examination 2012-13. It is a comprehensive work carried out under my supervision and to the best of my satisfaction. DATED : Mrs Lois Varghese PLACE : PGT (Biology) JNV, Thenzawl

4. ACKNOWLEDGEMENT At the first instant, we would acknowledge Lord Almighty for His incessant eternal support and encouragement. We always were brimming with enthusiasm with His divine blessings throughout the process of the completion of the project. We are deeply indebted to our guide and mentor Ms. Lois Varghese, PGT (Bio) for the constant help rendered by her. Her ever readiness to She was always with us in heart and soul during the progression of this work. Not underestimating, we are thankful to our peers for the support they lent to us. Last but not the least, we pay our fathomless gratitude to our parents for the mental zeal they bestowed on us. They whole-heartedly provided all the possible help to elevate this project to the highest level. PLACE : Ginngaihlian DATED: Roll No: PLACE: G R K C Avatar DATED: Roll No:

5. Principles and processes ofbiotechnology

6. DEFINITION • Biotechnology deals with techniques of using live organisms or their components or enzymes from organisms to produce products and processes useful to humans. • The European Federation of Biotechnology (EFB) furnishes its implication as: The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.

8. TECHNIQUES OF GENETIC ENGINEERING The technique of genetic engineering includes : • Creation of recombinant DNA • Use of gene cloning, and • Gene transfer. With this technique, we can isolate and introduce only the desired genes without introducing undesirable genes into target organisms.

9. WHAT IS CLONING? Cloning or making multiple copies of any template DNA needs that an alien DNA is linked with the origin of replication (a desired DNA sequence responsible for initiating replication) so that alien (foreign/desired) pieces of DNA can replicate and multiply itself in the host organism.

10. CREATION OF RECOMBINANT DNA • The first recombinant DNA was constructed by Stanley Cohen and Herbert Boyer in 1972 by; • Isolating the antibiotic resistance gene by cutting out a piece of DNAfrom a plasmid which was responsible for providing antibiotic resistance. The cutting of DNA at specific location was done by molecular scissors – restriction enzymes. • The cut pieces of DNA were then linked with the plasmid DNA. • The plasmid DNA acts as vector to transfer the pieces of DNA attached to it, to the recipient host bacterium. • Linking of antibiotic resistant gene (alien DNA) with plasmid DNA (vector) was possible with enzyme DNA ligase, which joins the cut ends of DNA molecules. • Hence, a new circular, autonomously replicating DNA created in vitro is formed called as recombinant DNA. • It replicates using the new host’s DNA polymerase enzyme and makes multiple copies. The ability to multiply copies of antibiotic resistant gene in E. coli is called cloning.

11. TOOLS OF RECOMBINANT DNA TECHNOLOGY • Restriction enzymes • Polymerase enzymes • Ligases • Vectors • Host organisms

12. RESTRICTION ENZYMES • Restriction enzymes belong to a class of enzymes called nucleases. They are of two types: (a) Exonucleases : Remove nucleotides from the ends/terminals of DNA. (b) Endonucleases : Cut the DNA at specific position of N-bases anywhere in the length except ends.

13. RESTRICTION ENDONUCLEASES-AN INTRODUCTION • Linn and Arber (1963) isolated two enzymes from E. coli responsible for restricting the growth of bacteriophage, one of them added methyl group (CH3 -) to DNA while the other cut the DNA into segments and is called restriction endonucleases. • Smith, Willoxand Kelley (1968) isolated and characterized the first restriction endonuclease from Haemophilus influenzae bacterium called it Hind-II. Hind-II always cut DNA at a particular site/point by recognizing a specific sequence of six base pairs called recognition sequence for Hind-II. Today more than 900 restriction enzymes have been isolated from over 200 strains of bacteria, each of which recognizes different recognition sequences.

14. RESTRICTION ENDONUCLEASES (contd) • The recognition sequence is palindromic where the sequence of base pairs reads the same on both the DNA strands when the orientation of reading is kept the same, i.e., 5’→3’ or 3’→5’ direction e.g. 5’- GAATTC- 3’ 3’- CTTAAG -5’. • Each RE function by ‘inspecting’ the length of a DNA and then binds to the DNA at recognition sequences. • RE cut the two strands of DNA double helix at specific points in their sugar-phosphate backbone, a little away from the centre of palindromic site, but between the same two bases on both the strands. As a result single stranded portions are left at the end of DNA in each strand called sticky ends. • Unwanted self ligation of vector DNA molecules by removing phosphate group from the 5’ end of a DNA molecule, leaving a free 5’ hydroxyl group, using alkaline phosphate from bacteria (BAP) or calf intestine (CAP). • These single stranded DNAs in each strand form hydrogen bonds with their complementary counterpart (single strands of alien DNA). This stickiness of the ends facilitates the action of enzyme DNA ligase. Hence RE are used in genetic engineering to form “Recombinant molecules of DNA”

15. RESTRICTION ENDONUCLEASES (contd) • When cut by the same RE, the resultant DNA fragments have the same kind of sticky ends and these can be joined together using DNA ligase.

18. NOMENCLATURE OF RESTRICTION ENZYMES • The first letter of the name comes from genus and the next two letters from the name of the species of the species of the bacterium (prokaryotic cell) from which they are isolated. • The next letter comes from the strain of the bacterium. • The Roman number following these four letters indicate the order in which enzymes were isolated from that strain of the bacterium, e.g. (a) Eco R-I is isolated from Escherichia coli RY 13 (b) Hind- II is from Haemophilus influenzae (c) Bam H-I is from Bacillus amyloliquefaciens (d) Sal-II is from Streptomyces albus.

19. CLONING VECTOR • Cloning Vector includes plasmid and bacteriophages, which have the ability to replicate within bacterial cells and independent of the control of chromosomal DNA. A vector should have very high copy number of their genome within their bacterial cell so that a linked alien piece of DNA, can multiply its number equal to the copy number by vector employed. • Vectors used at present are engineered in such a way that they help easy linking of foreign/alien DNA and selection of recombinant from non-recombinant.

20. CLONING VECTOR

21. FEATURES OF VECTOR • Following are the features that are required to facilitate cloning in a vector. 1. Origin of replication 2. Selectable marker 3. Cloning site (Recognisation vector) 4. Small size of vector • Origin of replication (ori): This is the sequence from where replication starts and a piece of DNA linked to this sequence can be made to replicate within host cells. This sequence also controls the copy number of vector DNA or linked alien DNA. • Selectable marker : It helps in identifying and eliminating non-transformants and help in selecting those host cells which contain the vector, i.e., transformants. • Cloning sites : The vector should have a few or atleast one unique recognition site, to link the foreign/alien DNA( to be use by RE). presence of one recognition site is preferable . the ligation of a alien DNA is carried out at restriction site present in one of the two antibiotic resistant genes.

22. VECTORS FOR CLONING GENES IN PLANTS AND ANIMALS • In plants, the Ti-plasmid (Tumour inducing plasmid) of bacterium Agrobacteriumtumefaciens has been modified (does not cause tumour ) is used as a cloning vector . •  Similarly, retroviruses also make the normal animal cells into cancerous cells. The retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells. • Once a gene or a DNA fragment has been ligated into a vector, it is transferred into a bacterial, plant or animal host.

23. PROCESSES OF RECOMBINANT DNA TECHNOLOGY IT INVOLVES THE FOLLOWING STEPS : • Isolation of DNA • Fragmentation of DNA by REs • Isolation of desired DNA fragment • Amplification of the gene of interest • Ligation of DNA fragment into vector • Transferring the recombinant DNA into host • Culturing the host cells in a culture medium at large scale • Extraction of desired products.

24. PROCESSES OF RECOMBINANT DNA TECHNOLOGYIn Detail 

25. ISOLATION OF DNA DNA is enclosed by membranes along with other macromolecules such as RNA, proteins polysaccharides and lipids. (a) The membrane is dissolved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cell), chitinase (fungus). (b) Long DNA molecules are interwined with histone proteins. RNA can be removed by treating with ribonuclease whereas the proteins can be removed by proteases. (c) Other molecules can be removed by treating by appropriate treatments and now purified DNA precipitates out after the addition of chilled ethanol. (d) It is seen as collection of fine thread in the suspension.

26. DNA that separates out can be removed by spooling

27. CUTTING DNA AT SPECIFIC LOCATION (a) Fragmentation of DNA is carried out by incubating purified DNA molecules with restriction enzyme at optimal conditions of temperature and pH for that specific enzyme. (b) Agarose gel electrophoresis technique is employed to check the progression of restriction enzyme digestion. (c) The similar process is repeated with vector DNA.

28. 3. SEPARATION AND ISOLATION OF DNA FRAGMENTS (a) DNA fragments being negatively charged, can be separated by forcing them to move towards anode under an electric field through a medium/matrix (agarose). The smaller the fragment size, the farther it moves. (b) DNA fragments can be visualized by staining by staining DNA with ethidium bromide followed by exposure to UV radiations. Bright orange colour bands in DNA became prominent in the gel. The separated bands of DNA are cut out from gel and extracted from the gel piece. This step is known as elution. (c) Purified DNA fragments are used for reconstructing recombinant DNA by joining them with cloning vectors.

29. 4. AMPLIFICATION OF GENES OF INTEREST USING PCR • PCR stands for polymerase chain reaction. In this reaction, multiple copies of gene /DNA of interest are synthesised in vitro using two sets of primers. • The enzyme DNA polymerase. • The enzyme extends the primer using the nucleotides provided in the reaction and the genomic DNA as template. • Continued DNA replication of segment of DNA can be amplified to approximately 1 billion times i.e., 1 billion copies are made. • Repeated replication is possible by the use of thermostable DNA polymerase (isolated from bacterium Thermusaquaticus) which remains active during high temperature induced denaturation o double stranded DNA. • Amplified fragment can be used to ligate with vector for further cloning.

31. JOINING OF DNAs (a) After cutting DNA with specific RE, the cut out gene of interest from the source DNA and the cut vector with space are mixed and ligase is added. (b) This results in the preparation of recombinant DNA. • INSERTION OF RECOMBINANT DNA INTO HOST CELL/ORGANISM There are several methods of introducing the ligated DNA into recipient cells. Recipient cells after making them ‘competent’ to receive, take up DNA present in its surrounding. So, if a recombinant DNA bearing gene for resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli cells, the host cells become transformed into ampicillin-resistant cells. If we spread the transformed cells on agar plates containing ampicillin, only transformants will grow, untransformed recipient cells will die. Since, due to ampicillin resistance gene, one is able to select a transformed cell in the presence of ampicillin. The ampicillin resistance gene in this case is called a selectable marker.

32. 7. OBTAINING THE FOREIGN GENE PRODUCT Ultimate aim of all recombinant technologies is to produce a desirable proteins by expression of recombinant DNA. The foreign gene gets expressed under suitable conditions, by culturing the host cell on a suitable medium.

33. RECOMBINANT PROTEIN Recombinant protein is produced if any protein encoding gene is expressed in heterologous host. (i) The transformed cells containing cloned gene of interest are grown in cultures

34. BIOREACTORS • Bioreactors are used for processing large volume of culture for obtaining the product of interest in large quantities. In bioreactors the raw materials are converted into specific products, e.g., enzymes. • A bioreactor provides the optimal conditions for achieving the desired product by providing optimal growth condition such as: (i) temperature (ii) pH (iii) substrate (iv) salt (v) vitamins (vi) oxygen

35. BIOREACTORS

36. APPLICATIONS OF BIOTECHNOLOGY

37. REASEARCH AREAS IN BIOTECHNOLOGY Three critical research areas of biotechnology are : (i) Providing the best catalyst in the form of improved organism usually a microbe or pure enzyme. (ii) Creating optimal conditions through engineering for a catalyst to act and (iii) Downstream processing technologies to purify the protein/organic compound.

38. BIOTECHNOLOGICAL APPLICATIONS IN AGRICULTURE Three options for increasing food production : • Agrochemical based agriculture • Organic agriculture • Genetically engineered crop based agriculture. Green Revolution has succeeded in tripling the yield of crops due to : • Improved crop varieties • Use of better management practices • Use of agrochemicals, i.e. fertilisers, insecticides and pesticides etc.

39. GENETICALLY MODIFIED ORGANISMS(GMO) GM plants are useful in many ways. Some of their unique features are: • More tolerant to abiotic stresses such as cold, drought, etc. • Have reduced dependence on chemical pesticides (pest-resistant crops). • Reduction in post harvest losses. • Increased efficiency of mineral usage by plants. • Enhanced nutritional value of food, e.g. Golden rice and sweet potato. • Used to create tailor made plants to supply alternative sources to industries in the form of starches, fuels and pharmaceuticals. • Production of pest-resistant plants, e.g. Bt-cotton, Bt-corn, rice and soyabean etc.

40. PEST - RESISTANT PLANTS Several nematodes parasitise a wide variety of plants and animals including human beings. • A nematode Meloidegyne incognitiainfects the roots of tobacco plants and causes a great reduction in yield. • A novel strategy was adopted to prevent this infestation which was based on the process of RNA interference (RNAi). RNAi takes place in all eukaryotic organisms as a method of cellular defence. This method involves silencing of a specific mRNA due to a complementary dsRNA molecule that binds to and prevents translation of the mRNA (silencing). • The source of this complementary RNA could be from an infection by viruses having RNA genomes or mobile genetic elements (transposons) that replicate via an RNA intermediate. • Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant . • The introduction of DNA was such that it produced both sense and anti-sense RNA in the host cells. • These two RNA’s being complementary to each other formed a double stranded (dsRNA) that initiated RNAi and thus, silenced the specific mRNA of the nematode. • The consequence was that the parasite could not survive in a transgenic host expressing specific interfering RNA. • The transgenic plant therefore got itself protected from the parasite

41. GENE THERAPY Gene therapy is a collection of methods that allow correction of a gene defect that has been diagnosed in a child/embryo required a mechanism to switch off a defective gene and to substitute a healthy gene copy. • The first clinical gene therapy was given in 1990 to a 4-year old girl with adenosine deaminase (ADA) deficiency. This enzyme is crucial for the immune system to function. • The disorder is caused due to the deletion of the gene for adenosine deaminase. • In some children ADA deficiency can be cured by bone marrow transplantation; in others it can be treated by enzyme replacement therapy, in which functional ADA is given to the patient by injection. • As a first step towards gene therapy, lymphocytes from the blood of the patient are grown in a culture outside the body. • A functional ADA cDNA (using a retroviral vector) is then introduced into these lymphocytes, which are subsequently returned to the patient. However, as these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. • However, if the gene isolate from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure.

42. MOLECULAR DIAGNOSIS Recombinant DNA Technology, polymerase chain reaction (PCR), and Enzyme-linked Immunosorbent Assay (ELISA) are some of techniques diagnosis because : • Very low concentration of bacteria or virus (before appearance of visible symptoms of disease) can be detected by amplification of their nucleic acids. • PCR is now used to detect HIV in suspected AIDS patients. • A probe is a piece of single stranded DNA that is tagged with a radioactive molecule and it is used to find it complementary by hybridization. • Presence of normal or mutated gene can be detected by this method .

43. TRANSGENIC ANIMALS Transgenic animals are those that have had their DNA manipulated to posses and express an extra or foreign gene, example transgenic rats, rabbits, pigs, sheep, cow, etc. Reasons for genetical modification of animals 1. Normal physiology and development 2. Study of disease 3. Biological products 4. Vaccine safety 5. Chemical safety testing

44. Modification of living organisms by human without regulation and some ethical standards cannot be allowed. • The modifications and uses of living organisms for public service have resulted in problems with the granting of patents for them. • Hence, Govt. of India has set up organization such as GEAC (Genetic Engineering Approval Committee) which will make decisions regarding the validity of GM research and the safety of introducing GM organisms for public service. ETHICAL ISSUES

45. BIOPIRACY Biopiracy is the term used to refer to the use of bioresources by MNCs and other organizations without proper authorization from the country and people concerned without compensatory payment. (i) Financially rich industrialized nations but poor in biodiversity exploit the bioresources and traditional knowledge of developing and underdeveloped poor countries rich in biodiversity. (ii) Some nations are developing laws to prevent such unauthorized exploitation of their bioresources and traditional knowledge.

46. SOME WONDERFUL FACTS mouse with human ear