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BIOTECHNOLOGY- principles and processes

Biotechnology ppt

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  • 1. BIOTECHNOLOGY- principles and processes

2. Introduction to biotechnology Definition: Biotechnology is the use of living systems and organisms to develop or make useful products, or "any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use European Federation of Biotechnology (EFB) has defined biotechnology as The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services. 3. Oldest form of biotechnology 4. Application of fermentation in production of wine and other alcoholic beverages is also a biotechnological technique 5. Principles of biotechnology Two important technique which enable development of modern biotechnology: 1. Alteration of chemistry of DNA & RNA to introduce into host organism to change phenotype of host- Genetic engineering 2. Maintenance of sterile ambience to enable growth of desired microbe/ eukaryotic cell in large quantities for manufacture of biotechnological products like vaccine, enzymes, beverages, drugs etc.- Chemical engineering 6. Asexual reproduction in organism preserves genetic information Sexual reproduction (hybridization) leads to variation- includes undesirable gene with desirable gene Genetic engineering- isolate & introduce only one or set of desirable genes without introducing undesirable genes in target organism Techniques of genetic engineering- creation of recombinant DNA, use of gene cloning & gene transfer to host Recombinant DNA (rDNA)/ alien DNA- cannot multiply itself until integrated in host genome When inherited in host DNA- ability to replicate due to origin of replication (host DNA)- initiates replication Alien DNA- linked with host DNA replicates & multiply itself along with host DNA- Cloning 7. Gene transfer to host require Vector Commonly used vector- Plasmid (small, circular, double stranded, self replicating extra chromosomal material of bacteria) First recombinant DNA was constructed- Stanley Cohen & Herbert Boyer (1972) by linking gene encoding for antibiotic resistance with plasmid of Salmonella typhimurium Isolation of desirable gene (antibiotic resistant)- cutting out piece of DNA from a plasmid responsible of antibiotic resistance which involve molecular scissors- restriction enzymes Desirable gene/ alien DNA linked with plasmid (vector) to transfer into host organism Linking of DNA involves DNA ligase- acts on cut DNA molecules & join their ends- new combination circular autonomously replicating DNA created in vitro- Recombinant DNA 8. rDNA transferred into Escherichia coli- DNA replicate using hosts DNA polymerase- multiple copies of antibiotic resistant gene- Cloning & E. coli will become antibiotic resistant Basic steps in genetically modifying organism: 1. Identification of DNA with desirable gene 2. Introduction of the identified DNA into host 3. Maintenance of introduced DNA in the host and transfer of the DNA into its progeny 9. Steps to genetically modify organisms 10. Basic steps involved in process Isolating Genomic DNA Fragmenting DNA Screening DNA fragments Insertion of DNA in vector Introducing DNA in host Culturing the cells Transformation of host cell 11. Basic steps involved in process Isolating genomic DNA Isolating genomic DNA from the donor. Fragmenting this DNA Fragmenting this DNA using molecular scissors. 12. Basic steps involved in process Insertion of DNA in a vector Screening the fragments Screening the fragments for a desired gene. Inserting the fragments with the desired gene in a cloning vector. 13. Basic steps involved in process Introducing in Host Culturing the cells Transformation of host cell Introducing the recombinant vector into a competent host cell Culturing these cells to obtain multiple copies or clones of desired DNA fragments Using these copies to transform suitable host cells so as to express the desired gene. 14. Biotechnology led to production of many products and provides many services for human welfare. 15. Tools od recombinant dna technology Recombinant DNA technology is accomplished with tools: 1. Restriction Enzymes: Restriction endonuclease & Restriction exonuclease 2. Polymerase enzymes 3. Ligase 4. Vectors 5. Host 16. Restriction enzymes Enzymes which is used to make cut in DNA in recombinant DNA technology 1963 two enzymes were isolated from Escherichia coli which restricts the growth of bacteriophage One enzyme added methyl group to DNA & other cut DNA and was named Restriction endonuclease Function of Restriction endonuclease depend on specific DNA nucleotide sequence First endonuclease isolated was Hind II; studies proved that Hind II always cut DNA at particular point by recognizing a specific sequence of six base pairs- recognition sequence of enzyme 900 restriction enzymes isolated from 230 strains of bacteria 17. Restriction enzymes belongs to nuclease class of enzymes. Its of two types: 1. Restriction exonuclease removes nucleotides from the end of DNA 2. Restriction endonuclease cut at specific positions within DNA While naming restriction enzymes- first letter represent genomic name & second two letter represent species name of the prokaryotic cell from which the enzyme was isolated Eg.- EcoRI- isolated from Escherichia coli RY 13; R- name of strain, I order in which the enzyme were isolated from that strain of bacteria 18. How restriction endonuclease act?? R.E inspects DNA sequence to find its specific recognition site to bind & cut specifically each of the two strand of the double helix at their sugar phosphate backbone R.E recognizes specific palindromic nucleotide sequence in DNA Palindrome in DNA- sequence of base pairs that reads the same on two strands when orientation of reading is kept the same, i.e., 5 3 in both strands 19. R. E cut the strand away from the center of palindrome sites but between same two bases on the opposite strand leading to formation of single stranded portion at the ends- Sticky ends Sticky end- hydrogen bond with their complementary cut counterparts- DNA ligase 20. Application of restriction endonuclease Used in the field of genetic engineering to form recombinant molecules of DNA- composition of DNA from different sources Same R.E (vector & source DNA)- same kind of sticky ends which can be joined by DNA ligase 21. Diagrammatic representation of recombinant dna technology 22. Separation and isolation of dna fragments Action of R. E result fragments of DNA- separated by Gel Electrophoresis Depends on negative nature of DNA External electrical field is applied to the medium- DNA move to anode Medium/ matrix used is agarose gel- natural polymer, extracted from sea weed Medium- acts as sieve & separate DNA according to their sizes; smaller fragments- farther Separated DNA- stained with Ethidium bromide & exposed to UV radiations- bright orange coloured bands of DNA in gel Separated bands of DNA- cut out from agarose gel & extracted from gel piece- Elution Purified DNA fragments- constructing recombinant DNA by joining them with cloning vector 23. Electrophoresis- separation of DNA fragments based on size Staining- Ethidium bromide Exposure to UV radiation Cutting out DNA fragments 24. Cloning vectors A cloning vector is a small piece of DNA, taken from a virus or a plasmid of bacteria, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes Common vectors- Plasmids & Bacteriophage Salient features of cloning vectors: 1) Ability to replicate within bacterial cell independent of chromosomal DNA 2) High copy number per cell; Plasmids- 15- 100 copies per cell 3) Alien DNA when integrated with vector can multiply equal to copy number of vector 4) Vectors can be engineered for easy linking of foreign DNA & selection of recombinant from non- recombinants 25. Features to facilitate cloning into vectors Features required to facilitate cloning into vectors: 1. Origin of replication (ori) 2. Selectable marker 3. Cloning sites 4. Vectors for cloning genes in plants & animals 26. 1. Origin of replication (ori): Sequence on DNA where replication starts/ initiates Alien DNA when linked to it- replicate within host cell Regulates & control copy number of linked DNA/ alien DNA/ target DNA If multiple copies of target DNA required, then target DNA be cloned in a vector whose origin of replication supports high number 2. Selectable marker: Identify & eliminate non- transformants from transformants & selectively permit growth of transformants Transformation- procedure through which rRNA introduced into host bacterium- change in phenotype Eg.- E. coil- resistance against ampicillin, chloramphenicol, tetracycline or kanamycin- selectable markers which is lacked in normal E. coli 27. 3. Cloning sites Region in the vector where ligation of target DNA takes place Created by commonly used specific R. E at the recognition sites/ restriction sites within a vector where ligation of alien DNA takes place pBR322- genetically engineered plasmid; widely used E. coli cloning vectors; the ampR gene- encodes for ampicillin resistance protein, and the tetR gene- encodes for tetracycline resistance protein; have specific restriction sites for different Restriction endonucleases (BamH I, Sal I, Hind III, Cla I, EcoR I, Pvu I Pvu II, Pst I) Presence of more than one recognition site within vector will complicate gene cloning Ligation of alien DNA- restriction site present in one of two antibiotic resistance genes Inactivate the gene- enable to choose transformants from non transformants 28. If alien DNA is ligated at Bam HI site in tetracycline resistance gene- recombinant DNA loose its resistance against Tetracycline (transformants/ recombinants) Transformants can be selected from non transformants- plating bacteria on ampicillin containing medium- bacteria grows Transformants growing in ampicillin transferred to tetracycline medium- transformants cannot grow & non recombinants grow- due to gene gets inactivated- insertion 29. Selection of recombinants due inactivation of antibiotics- cumbersome process due to simultaneous plating on two different antibiotics Alternative selectable marker developed- differentiates to produce colour in the presence of chromogenic substance Recombinant DNA sequence is inserted within coding sequence of enzyme - galactosidase- inactivation of enzyme- insertional inactivation Presence of chromogenic substrate (- galactosidase)- blue coloured colonies (bacteria with plasmid with no insert) Presence of insert- inactivation of - galactosidase- colonies do not produce any colour- identify recombinant colony 30. 4. Vectors for cloning genes in plants & animals: Bacteria & virus- transform eukaryotic cell by delivering genes Agrobacterium tumifaciens (pathogen of dicot plants)- deliver a DNA piece- T- DNA & transform normal cell to tumor which produces chemicals required by the pathogen Retrovirus- transforms normal animal cell to cancerous cell An understanding of mechanism of delivering genes by pathogens to their host can help to understand the tools of pathogen & can be used to deliver genes of interest in human Tumor inducing plasmid (Ti) of Agrobacterium tumifaciens- modified into cloning vector- non pathogenic to plants & have ability to deliver genes of interest to variety of plants Retrovirus- disarmed; delivers genes of interest in animals When gene of interest is ligated into a suitable vector- transferred into suitable host (bacteria, plant or animal)- multiply 31. Plant vector- Ti plasmid 32. Animal vector 33. Competent host (for transformation with rdna) rDNA should enter cell to transform cells But DNA cannot enter cell in a medium without treatment- hydrophilic Host should be made competent to take up DNA (vector) Different techniques to make host competent: 1.Chemical Treatment: Treating hosts (bacteria) with specific concentration of a divalent cation (Ca)- increases efficiency & make DNA enter through cell wall of host Heat shock (sudden change in temperature)- mixture of rDNA & host cells Incubation of host & rDNA on ice & then placing them at 42C & then back to ice Enable bacteria to take up rDNA 34. 2. Microinjection: rDNA directly injected into nucleus of host cell Common for animal cell 3. Biolistics & gene gun: Host cell bombarded with high velocity micro- particles of gold or tungsten Micro- particles are coated with recombinant DNA Used for plant cell 4. Disarming pathogen: Pathogenicity of vector is removed- disarmed pathogens Disarmed pathogen vector- allowed to infect host & transfer r DNA 35. Techniques to introduce recombinant dna 36. Process of recombinant DNA technology Recombinant DNA technology involves following steps: 1. Isolation of genetic material DNA 2. Fragmentation of DNA by restriction endonuclease 3. Isolation of desired DNA fragments 4. Ligation of the DNA fragments into a vector 5. Transferring rDNA into host 6. Culturing host cells in a medium at large scale 7. Extraction of desired product 37. 1. Isolation of Genetic material (DNA) Most of eukaryotic cells have DNA as genetic material cells To form rDNA, the DNA should be pure, i.e., free from all biomolecules DNA- located inside nucleus enclosed by plasma membrane, genes interwined with histone protein Cell have to break open to release DNA & other macro molecules (RNA, proteins, polysaccharides & lipids) Cells are lysed by lytic enzymes: lysozyme (bacteria), cellulase (plant cell), chitinase (fungus) Macromolecules- treated with specific enzymes to remove; RNA- ribonuclease, Proteins- Protease, Purified DNA precipitates with addition of Ethanol- appear as collection of fine threads in suspension 38. Steps for dna extraction 39. 2. Cutting of DNA at specific location Purified DNA (source DNA/ desirable gene DNA & vector DNA) incubated with specified restriction enzymes for the process- Digestion at optimal condition Progression of restriction enzyme digestion is checked- agarose gel electrophoresis (source DNA & vector DNA) DNA- negatively charged so move towards positive electrode (anode) Once gene of interest & cut vector with space for gene of interest is isolated, DNA ligase is added This step is a preparation of recombinant DNA 40. 3. Amplification of Gene of Interest using PCR PCR (Polymerase Chain Reaction)- technology used to amplify a single or a few copies of a piece of DNA to generate thousands to millions of copies of a particular DNA sequence in vitro. Components required- Primers (small chemically synthesized oligonucleotides that are complementary to the regions of DNA), Nucleotides & DNA polymerase- incubated together Steps in PCR 1. Denaturation of double stranded DNA by heating- single stranded DNA 2. Primer is added- binds to complementary region- Annealing 3. DNA polymerase- polymerization with nucleotides in medium; genomic DNA act as template 4. Replication repeated many times- 1 billion copies of desirable DNA- Amplification & involve thermostable DNA polymerase- Thermus aquaticans- to facilitate replication even at high temperature (denaturation) 41. Polymerase chain reaction 42. 4. Insertion of Recombinant DNA into Host cell/ organism Insertion of rDNA possible by making host competent Achieved by- Chemical treatment, microinjection, biolistic & gene gun method or disarming pathogen Phenotype of organism alters when rDNA is transferred to host Eg.- rDNA with gene resistance against antibiotic Ampicillin transferred to E. coli (host)- phenotype alters; resistant against ampicillin- transformants When plated on agar plate with ampicillin- transformed will grow (change in phenotype) & non- transformed- die Presence of ampicillin resistance gene- select transformed cells & considered as Selectable marker 43. 5. Obtaining the Foreign gene product Alien DNA binds with cloning vector- rDNA, transferred to suitable host (bacteria, plant or animal cell) rDNA- expresses itself (target protein) under appropriate optimal conditions & after cloning of rDNA to particular number of copies Target protein- produce in large scale In heterologous host if protein encoding gene is expressed- recombinant protein Heterologous host- gene or gene fragment that does not naturally present in host & the gene expresses itself (recombinant protein) Cells which harbor cloned genes of interest- grown in laboratory in small scale Culture of dividing host- used to extract desired protein & then purified by different separation techniques 44. In large scale cells can be cultured in continuous culture system Here the used medium is drained out form one side & fresh medium added from other to maintain the cells- physiologically most active log/ exponential phase This culture method produce a larger biomass leading to higher yields of desired protein Biomass- biological material derived from living, or recently living organisms To produce large quantities of products- Bioreactors are employed (100- 1000 liters)- large volumes of culture is processed Bioreactors- vessels in which raw materials (recombinant organism like microbial plant, animal or human cell & culture medium with ambient condition)- biologically converted into specific products (protein/ enzymes) Optimal growth is achieved with growth conditions- temperature, pH, substrate, salts, vitamins & oxygen 45. Structure of Bioreactors: Commonly used- stirring type Cylindrical & with curved base to facilitate mixing contents Equipped with stirrer- even mixing & oxygen availability throughout reactor Oxygen delivery system- bubble air through reactor Agitator- thorough mixing of contents Also have foam control system, temperature control system, pH control system & sampling port Sampling port- withdraw small volumes of culture periodically to check the progression 46. bioreactors 47. 6. Downstream Processing Downstream processing refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal or plant tissue or fermentation broth Products obtained from bioreactors- subjected to series of process before marketing as a finished product Process include separation & purification of biological products Products are formulated with suitable preservatives & then should undergo thorough clinical trials like drugs Strict quality control testing is required for the final biological products