Biotechnology (As per Karnataka PUC Syllabus) Dr M. Jayakara Bhandary Associate Professor of Botany Government Arts and Science College, Karwar Karnataka email@example.comIntroduction: • Biotechnology is any technique that uses living organisms or parts of organisms to make or modify products, to improve plants or animals, or to develop micro organisms for specific uses, OR It is the application of scientific and engineering principles to the processing of materials by biological agents to provide goods and services. Scope of biotechnology: Field of Applications/Activities Application AgricultureNew varieties of crop plants with improved characteristics like pest resistance, stress tolerance, high yield, herbicide resistance, better nutritional quality etc. can be produced by combining Recombinant DNA Technology and Tissue Culture Technique. Such genetically modified plants are called transgenic plants. Elite Elite and disease resistant plants can be propagated by micropropagation. Plants can be Genetically Engineered to produce proteins of commercial importance like vaccines, enzymes, drugs, biodegradable plastic, etc Animal New Breeds of domesticated animals can be produced by combining husbandry Recombinant DNA Technology and cloning technique. eg. Fast growing sheeps, pigs, etc. Mammals like sheep and cattle can be genetically designed to produce proteins and medicines of importance in their milk. Obtaining pharmaceutical products from genetically modified animals is called biopharming. Medicine l Drugs, vaccines etc. can be produced by microorganisms, plants or animals through genetic engineering. eg. insulin production by bacteria. Genetic diseases can be treated by replacing the defective disease causing genes with healthy genes. This method is called gene therapy. Traditional vaccines can be replaced by directly introducing DNA molecules coding for vaccines into the body. Such DNA are called DNA vaccines. Diagnostic method and kits for early detection of genetic diseases, accurate and faster detection of pathogens in the body tissues etc. can be developed. eg. Monoclonal antibodies, Enzyme linked immunosorbant assay (ELISA), Radio immuno assay (RIA), etc. Pollution Biotechnological methods can help in dealing many environmental management problems. eg. microbes can be developed and used as indicators of different types of pollution. Microbes can be used to degrade pollutants, in the treatment of sewage, industrial wastes, etc.( Bioremediation) Industry Micro organisms can be genetically designed to efficiently undertake various industrial processes. eg. Lignin degrading microbes are used in paper industry to digest the pulp.
Microbes are used to purify metals like gold, copper etc. from their ores. This method is called biomining. Fermenters, bioreactors etc. developed on biotechnological principles play important roles in various industries like paper, pharmaceutical, wine industry, etc.Forensic Biotechnological tool like genetic finger printing is used in forensicScience science as an judicially approved method to identify criminals, solve cases of parental disputes, etcBiodiversity Methods like tissue culture, protoplast culture, embryo transfer andconservation animal cloning can be used for the conservation of rare plants and animalsBasic Biotechnological tool like gene cloning provides efficient method to studyscientific genetic processes like gene expression, regulation, disease development etc.research It is also used in genome mapping projects in which entire genome (set of genes of an organism) is sequenced and documented. eg. Human genome projectComputer Ccomputers are efficiently used to analyse and store the enormousScience information generated by biotechnological experiments. This new area which is the combination of IT with BT is called Bioinformatics. Chips called biochips made from different biological materials may replace the silicon chips used in computer.Genetic Engineering (GE): • Genetic engineering refers to genetically modifying organisms by transferring genes from other organisms, using the recombinant DNA technology. • Organisms with new combination of genes that are produced by genetic engineering are called transgenic organisms or Genetically Modified Organisms (GMO’s).Tools used in Genetic Engineering:Vectors: • Vectors are DNA/RNA molecules used to transport foreign genes into a recipient cell or organism, in genetic engineering experiments. Vector should replicate the transferred gene inside host cells. • Shuttle vectors are those which replicate the foreign gene both in prokaryotic and eukaryotic host cells. Ex. Yeast episomal plasmids. • Expression vectors are capable of replicating and expressing (protein production) the foreign gene inside host cells. They have additional promoter and terminator sequences for transcription. • Different types of vectors are listed below: Types of vector Desription/use Example Plasmids Extrachromosomal, self replicating , pSC 101, pBR 322, pUC 18, double stranded, circular DNA molecules etc. found in bacterial and yeast cells. Yeast plasmids Presently, only artificial designed plasmids are used as vectors. Ti (Tumor inducing) plasmid of the Ti plasmid, Ri plasmid bacterium Agrobacterium tumifaciens & Ri plasmid of A. rhizogenes are used to
transfer genes to plant cells. Plant viruses Carry genes to plant cells Caulimo, Gemini and Tobamo viruses Animal viruses Carry genes to animal cells, especially Retroviruses, Adenoviruses human cells for gene therapy Bacteriophages Viruses which infect bacterial cells Phage lambda, M13 Cosmids Combination of plasmid DNA and ‘Cos’ - (cohesive) regions of phage lambda. Produced by Collins & Hohn, 1978 Phagemids Combination of plasmid DNA and single stranded DNA segment of phage M13. Artificial Artificial Chromosomes designed to carry Yeast Artificial chromosomes large-sized eukaryotic genes Chromosome(YAC), Human artificial chromosomes (HAC)Enzymes: • Enzymatic cutting and joining of DNA is important step in genetic engineering. • Restriction enzymes (REN) are enzymes which cut DNA at specific palindromic sequences. They are called DNA scissors. They occur naturally in bacterial cells. • A palindrome is a DNA sequence of 4-8 bases which reads the same in both the strands, in a particular direction (5’ to 3’ or vice -versa). Each REN identify a specific palindromic sequence. • REN are of two types: Type I identify palindromic sequences but cut the DNA strands outside it. They are not of any use in GE., • Type II restriction enzymes are commonly used in genetic engineering because they cut the DNA within the palindromic sequences, producing either blunt ends or sticky ends. Some important type II restriction enzymes: Name of REN Source organism Target palindromic sequence Eco R1 Escherichia coli GAATTC/CTTAAG Eco RII Escherichia coli GGATCC/CCTAGG Hind III Haemophilus influenzae AAGCTT/TTCGAA Hpa II Haemophilus parainfluenzae CCGG/GGCC Sal I Streptomyces albus GTCGAC/CAGCTG • DNA ligases are enzymes which join or ligate the sugar-phosphate backbones of two DNA fragments by forming phosphodiester bond between them. They act like DNA stitches. Activity of DNA ligase was first demonstrated by Mertz and Davis in 1972. T4 DNA ligase obtained from T4 bacteriophage is commonly used. • Other enzymes useful in GE are listed below: Enzyme Application Alkaline phosphatase Remove PO4 group from 5’ end of DNA or RNA Reverse transcriptase Synthesis of single stranded complementary DNA (cDNA) from RNA
DNA polymerase I Convert single stranded cDNA into double stranded cDNA Taq polymerase Used in Polymerase Chain Reaction to clone DNA Terminal transferase Adds nucleotides to 3’ end of DNA or RNA• Bioreactors are large vessels specially designed to grow microorganisms, plant or animal cells in a large scale and to harvest the commercially valuable product which they produce.• E. coli is the common host used in Genetic Engineering.Recombinant DNA Technology:• Recombinant DNA is artificially created new combination of DNA molecules by combining DNA segments taken from two or more different organisms or sources. First successful recombinant DNA molecule was produced by Stanley Cohen and Herbert Boyer in 1973.• The method of producing many identical copies of a gene is called gene cloning. Earlier, gene cloning was done by transferring the gene into a bacterial cell and multiplying the cell. Now, the same can be easily carried out by machines called Thermocyclers which uses Polymerase Chain Reaction (PCR) to produce millions of copies of a gene, in a few hours.• PCR technology was developed by Kary Mullis in 1985. It uses a special heat tolerant DNA polymerase called Taq polymerase isolated from a thermophilic bacterium called Thermus aquaticus.• Construction of recombinant DNA involves mainly cutting DNA fragments by restriction enzymes and rejoining them by DNA ligase. The important steps involved are given below: Steps Details Obtaining the The DNA molecule or gene of interest can be obtained mainly by two gene of sources, as follows: interest a) from gene libraries : Gene libraries are collections of natural copies of genes or complementary DNA (cDNA) copies of genes (cDNA is obtained by reverse transcription of mRNA by using reverse transcriptase enzyme). b) Synthetic DNA : DNA segments coding for specific polypeptides can be made with the help of DNA synthesis machines. Vector A suitable vector DNA molecule is selected based on the objective of the selection experiment Restriction The DNA molecule having the gene and the vector molecule are cut with digestion the same appropriate restriction endonuclease enzyme to generate DNA fragment with sticky ends. Ligation or The gene and vector DNA fragments are mixed together in the presence of gene splicing DNA ligase enzyme. Owing to the presence of complementary ends, the two fragments join together first by hydrogen bonding, followed by phosphodiester bond formation. The gene-vector complex thus formed is the recombinant DNA molecule. Transfer to The recombinant DNA molecule is transferred to a suitable host cell. Host host cell cells like E. coli cells are mixed with recombinant DNA and treated with calcium chloride followed by a mild heat shock. They absorb the recombinant DNA into their cells. The host cells which have successfully absorbed the recombinant DNA are selected and multiplied.
Extraction of The population of host cells can be then used for the purpose of the gene or gene experiment such as recovery of the cloned gene or extraction of the cloned product gene product (protein) or formation of transgenic organisms.Recombinant insulin production: • Recombinant DNA technology is used to obtain many copies of a gene or protein. Human insulin was the first protein produced by recombinant method by Eli Lilly Company in 1982. The commercially available recombinant insulin was called humulin or recosulin. • Functional insulin is a relatively small protein comprising two polypeptides - The A chain of 21 amino acids and the B chain of 30 amino acids. The two chains are interconnected by two disulfide bonds. • Insulin was originally identified by Frederick Banting & Howard Best in 1921. • In humans, insulin is synthesised as a precursor molecule called preproinsulin which actually has 4 polypeptide parts: A chain (21 amino acids), B chain (30 amino acids), C chain in between A and B (33 amino acids) and the N-terminal leader segment (16 amino acids). • The preproinsulin is processed after translation (post-translational modification) by removing the leader and C chains. The remaining A and B chain attach to each other by two disulphide bonds to form functional insulin. Preproinsulin without the leader segment is called proinsulin. • The original method followed in successfully engineering E. coli cells to synthesize human insulin is summarised as follows:Steps EventsArtificial gene Functional insulin protein consists of two sub-units of polypeptide chains calledsynthesis chain A and B. Synthetic genes coding for A and B polypeptide sub-units of human insulin were constructed by Itakura and his group in 1977.Splicing Each synthetic gene was separately inserted into the tail end of Lac Z gene (which produces the protein b-galactosidase) of pBR322 plasmid molecules to form recombinant DNA molecules. Such recombinant DNA molecules are capable of producing fusion proteins which contain both b-galactosidase and insulin sub-units together.Cloning The recombinant plasmids containing insulin A and B genes were inserted into separate E. coli cells. These modified E. coli cells were grown in large scales during which the fusion proteins were synthesised and accumulated in the host cells.Extraction & The fusion proteins were extracted from the host cells separately, purified andpurification of treated with cyanogen bromide to separate insulin sub-units from b-fusion proteins galactosidase protein.Formation of Purified A and B insulin sub-units thus obtained are finally mixed together tofunctional produce functional insulin molecules. In the functional insulin, A and B subunitsinsulin attach together by disulphide bonds
Some of the human proteins that have been synthesized from genes cloned in bacteria and / oreukaryotic cells:Protein Used in the treatment of Protein Used in the treatment ofInsulin Diabetes Epidermal growth Ulcers factorSomtostatin Growth disorders. Fibroblast growth Ulcers factorSomatrotropin Growth disorders Erythroprotein AnaemiaFactor VIII Haemophilia Tissue plasminogen Heart attack activatorFactor IX Christmas disease Superoxide dismutase Free radical damage in kidney transplantsInterferon -a Leukaemia and other Lung surfactant Respiratory distress cancers. proteinInterferon - b Cancers, AIDs g1-antirypsin EmphysemaInterferon -g Cancers, rheumatoid Serum albumin used as a plasma arthritis supplementInterleukins Cancers, immune Relaxin Used to aid childbirth. disordersGranulocyte colony Cancers Fibroblast growth Ulcersstimulating factor factorTumour necrosis Cancers Erythroprotein AnaemiafactorDNA fingerprinting: •DNA fingerprinting is a technique which helps in the establishment of identity of a person or any organism based on uniqueness of DNA. Alec Jeffreys developed this technique in 1985. • Presence of Variable Number Tandem Repeats (VNTR) in the genome is the basis of DNA finger printing. • Transferring the single stranded DNA bands from electrophoretic gel slab to a nitrocellulose sheet is called Southern blotting, in honour of Edwin Southern who developed this technique. • Similar transfer of protein bands is called Western blotting (developed by Towbin & group in 1979) and transfer of RNA to special type of cellulose paper is called Northern Blotting (developed by Alwine & group in 1979). • DNA probes are short single stranded DNA segments containing radioactive P32 which perfectly bind to specific complementary single strand DNA fragments. • DNA fingerprinting is useful in detection of criminals and paternity testing. Steps in DNA fingerprinting: DNA extraction DNA is extracted from the tissue samples such as blood, semen, cells, hair etc., collected from the crime spot and also of the various suspects. Restriction digestion Extracted double standard DNA samples are digested with restriction enzyme to form fragments of different length based on the pattern of VNTR’s and restriction sites. Electrophoretic The DNA fragments are separated according to their size into distinct separation bands on a agarose gel sheet by a method called gel electrophoresis.
Single strand The double strand DNA fragments on the gel sheet are separated into seperation single strands by treatment with an alkali (NaOH) solution Southern Blotting: The single stranded DNA bands are transferred from the gel slab to a nitrocellulose sheet by southern blotting, for visual observation Treating with DNA on the nitrocellulose sheet are treated with radioactive DNA radioactive probes probes. DNA probes presently used are complementary to the VNTR sequences of Human DNA which bind only to VNTR’s. Auto radiography The nitrocellulose sheet is X-ray photographed. Only such DNA bands to which radioactive probes bind appear as dark spots in the x-ray photograph which is called autoradiograph. The number, size, and location of final dark spots in the photograph is unique to a person like fingerprints. Therefore, such a DNA band pattern obtained in the autoradiograph is called DNA fingerprint. Comparison On visual or computer analysis, if the DNA fingerprint of any suspect matches with that of the DNA collected from the crime spot, it can be concluded that both DNA samples belong to the same individual.Gene Therapy: • Gene therapy is any procedure used to treat a disease by modifying or manipulating the genetic composition in the cells of the patient. • Gene therapy is tried to treat genetic diseases like Adenosine deaminase deficiency (ADA), Severe combined immunodeficiency (SCID) , Cystic Fibrosis (CF), Cancers, etc. Harmless viruses like adenoviruses, retroviruses, Lentiviruses etc are used as vectors in gene therapy experiments.Types of gene therapy:Type Description ExampleSomatic gene Only the somatic cells of the body are used as -therapy targets for gene transfer. The presently developed methods belongs to this type and the therapy is restricted only to the diseased tissue.Germline gene germ cells used for gene therapy. In this case, all -therapy the cells developing from the altered germ cell will carry the introduced gene and it will also be inherited to future generations.Ex vivo gene gene transfer is carried out with the affected cells Insertion of ADA gene totherapy which are isolated from the body of the patient and cultured bone marrow cells grown in cell cultures. The genetically modified cells are then reintroduced into the body of the patientIn vivo gene transfer of genes directly into the affected cells of Gene therapy for cystictherapy the body, without isolating them. fibrosis (CF) where CFTR genes are introduced directly to epithelial cells of respiratory tract.Diseases experimentally treated by gene therapy:Disease symptoms Cause Gene therapy
Cystic Thick and sticky A defective CFTR gene (Cystic Normal CFTR geneFibrosis mucus that clogs the Fibrosis Transmembrane Regulator introduced to the(CF) respiratory passages gene) produces a defective CTCR epithelial cells of the (Cystic transmembrane conductance respiratory tract by in regulator) protein which absorbs more vivo method using water from mucus, makes it thick. adenovirus vector. CFTR gene discovered by Fransis Collins & Lap chee Tsui in 1989 on Chromosome 7.ADA/ Absence of Adenosine Defective ADA gene present on Insertion of normalSCID deaminase enzyme, chromosome 20. ADA gene to cultured destruction of T- bone marrow cells lymphocytes, weak using retrovirus immune power. vectorCancers Uncontrolled cell Gene mutation, presence of tumor Insertion of Tumor division inducing genes, etc suppressing genes like P 53 or genes for Tumor Necrosis Factor (TNF)Monoclonal antibodies: • Pure antibodies of predetermined specificity which can react with only a specific type of antigen are called monoclonal antibodies (MAB). • Georges Kohler and Cesal Milstein discovered hybridoma technology for the production of monoclonal antibodies. • Hybridoma is a special type of hybrid cell formed by the fusion of antibody forming B- lymphocyte and bone-marrow tumour cell called myeloma. Poly ethylene glycol (PEG) is the fusogen which induces fusion of these cells. • MAB are widely used in: 1. Diagnostic techniques like ELISA (enzyme linked immuno sorbant assay) which are used for faster and accurate detection of pathogenic viruses and bacteria. 2. Immunopurification: Separation of a specific biological substance from a mixture of many similar substances. 3. Targeted therapy: Therapy against diseases like cancer in which only specific antigens need to be attacked with the help of specific antibodies. 4. Blood typing: The anti A and anti B sera used in detection of ABO blood groups are monoclonal antibodies produced against antigen A and B of RBC’s.Human genome project: • A genome is all the DNA in the haploid set of chromosomes of an organism, which makes up all its genes. This term was coined by H. Winkler in 1920. • Human genome project (HGP) aim was to sequence the entire human genome. It was undertaken by Human Genome Organisation (HUGO). It was begun in October 1990 in USA and completed in March 2003. • Important findings of Human Genome Project are : 1. Total number of functional gene in human cells are about 30,000 - much lower than the expected number of about 1 lakh.
2. The human genome has 3164.7 million nucleotide base pairs. 3. 99.9% of bases are exactly the same in all people. 4. Only 2% of the DNA actually code for proteins, the other 98% DNA function is unknown. This DNA with unknown function is called junk DNA. 5. Average size of human genes is 3000 bases. The largest human gene is dystrophin gene with 2.4 million bases. 6. Chromosome-1 has maximum number of genes (2968) and Y-chromosome has the lowest number of genes (231) 7. The functions of about 50% of the discovered genes are unknown.Improvement of Plants: • Plant breeding is the production of new varieties of plants with improved characteristics like better yield, better nutritional quality, disease resistance, fast growth etc. • Hybridisation is cross fertilising parental plants which genetically differ from each other. • Mutation breeding involves induction of mutations in plants by artificial methods like treating with chemicals and radiation. Treating plant propagules like seeds, seedlings or vegetative buds with chemicals like ethyl methyl sulfonate (EMS) or with ionising radiation like gamma rays produced by radioactive substances such as cobalt or caesium are the popularly used methods of inducing mutations. Sharbati Sonora is a variety of wheat produced by mutation breeding which was responsible for green revolution in India. • Polyploidy breeding involves creating new varieties of plants by inducing polyploids or multiple sets of chromosomes in plant cells. Polyploidy can be induced by a chemical called colchicine. • Plant tissue culture is a technique of growing isolated plant cells or tissues in an artificial nutrient medium, under aseptic conditions. • Each cell of any plant has got the inherent potential or ability to develop into a complete functional plant, when cultured under suitable conditions. This unique property of plant cells is called totipotency. • Callus is a mass of undifferentiated cells. Cells of the callus are induced to develop clusters of small plantlets by forming organs like shoots and roots. This step of organ formation is called organogenesis and is induced by adding plant hormones like auxins and cytokinins, in appropriate ratios. A high auxin and a low cytokinin concentration stimulates root formation. Low auxin and high cytokinin induces shoot formation. • Organ culture is culturing isolated plant organs under aseptic conditions in a nutrient medium where they continue to grow retaining their characteristic structures. Tisssue culture media and their constituents: • A medium provides all the necessary requirements for the multiplication and differentiation of cultured cells. Various media like Murashige & Skoog medium (MS medium), Whites medium, etc. are used for plant tissue culture. These media differ mainly in the quantity of the constituents included. The major constituents of a culture medium are: 1. Inorganic nutrients like carbon, hydrogen, oxygen and additional 12 essential elements (N, P, S, Ca, K, Mg, Fe, Mn, Cu, Zn, Band Mb). 2. Organic nutrients: They can be broadly classified into carbon sources (sucrose, glucose and fiuctose) and nitrogen sources (vitamins and amino acids). 3. Growth hormones: The growth hormones included are: a) auxins to induce cell division and root differentiation. Commonly used auxins are IBA (Indole 3-butyric acid ), NAA (Naphthalene acetic acid), 2, 4-D, etc.
b) cytokinins to facilitate cell division and shoot differentiation. BAP (Benzylamino purine) and kinetin are the commonly used cytokinins. c) Gibberellins like GA3 are added for special purposes like plantlet formation from somatic embryos.4. Agar: Agar is a polysaccharide obtained from seaweeds. It is added as a solidifying agent to culture media. Agar is not a nutrient.• Some common terms associated with plant tissue culture:Callus culture The explant is induced to form callus on a solid nutrient medium containing agar. The callus is then induced to produce. organs like shoots and roots (Organogenesis) or somatic embryos (Somatic embryogenesis), by manipulating the concentration of growth hormones.Somatic embryos These are embryo-like structures produced by cultured tissue. They germinate to form plantletsCell culture Culture of isolated cells usually in a liquid medium (medium without agar). Such a liquid culture is also called suspension culture.Protoplast culture Culture of plant protoplasts (cells without cell wall) isolated from plant tissue or cultured cells. Cell walls are digested by treating cells with enzymes like pectinases and cellulases.Haploid plants Plants produced by culturing haploid tissue like pollens or anther.Cytoplasmic Fusion oftwo protoplasts, usually derived from two differenthybridization plant sources, to form a hybrid cell which is called cytoplasmic hybrid or cybrid. Such cybrids carry the cytoplasm derived from both of the parental protoplasts and nucleus from one of the protoplasts. Fusion of protoplasts is induced by chemical agents like polyethylene glycol (PEG), Sodium nitrate, etc.Somoclonal This is the variation observed between the plants regeneratedvariation by tissue or cell culture• Genetically modified plants which contain one or more artificially transferred genes in their cells are called transgenic plants. The gene artificially transferred to an organism is called transgene. Genetic modification of eukaryotic cells (plant or animal) by transfer of foreign gene is called transfection. The process of producing transgenic organisms like plants and animals by transfection is called transgenesis.• Ti plasmid (Tumour inducing plasmid) of Agrobacterium tumefaciens and Ri plasmid of Agrobacterium rhizogenes are used as vectors in the production of transgenic plants.• Golden rice is a transgenic rice made by inserting three genes needed for producing beta carotene - a vitamin A precursor substance, by Ingo Potrykus and Peter Beyer in 1999.• These three genes are:1. psy gene coding for the enzyme phytoene synthase,2. lyc gene for lycopene cyclase. These two genes were from the daffodil plant (Narcissus psuedonarcissus).3. ctrl gene for an enzyme phytoene desaturase was from the soil bacterium Erwinia uredovora.
• Eating golden rice is believed to help in preventing diseases like xerophthalmia and blindness that are caused by vitamin A deficiency. • Pesticide resistant Bt gene is transferred to plants like corn, maize, etc from the bacterium Bacillus thuringensis. • Vaccines which are produced inside edible parts of transgenic plants are called edible vaccines.Improvement of animals: • Insemination of a female animal by methods other than natural mating is called artificial insemination. • Multiple or Superovulation is the production of more than normal number of eggs. Females that are donors of eggs are injected with hormones to stimulate increased egg production. Embryo transfer is removal of an embryo in its early stage of development from its own mother’s (the donor’s) reproductive tract and transferring it to another female’s (the recipient’s or surrogate mother) reproductive tract. The technique which combines both superovulation and embryo transfer is called Multiple Ovulation and Embryo Transfer (MOET). • A stem cell is an immature cell that has the potential to become specialized into different types of cells of the body of animals. Embryonic stem cells and Adult stem cells are the two types of stem cells. Stem cells are normally cultured on a layer of supporting cells called feeder layer. Cell cultures stored for a long time are called immortal cell lines. • Cloning for the purpose of embryonic stem cells to be used in tranplantation treatment is called therapeutic cloning. • Transgenic animals are animals that have been genetically engineered to contain one or more foreign genes in their cells. Ex. Transgenic cattle.