Presentation on theme: "Principles and Processes"— Presentation transcript:
1 Principles and Processes Chapter 11BIOTECHNOLOGYPrinciples and Processes
2 BIOTECHNOLOGYDeals with techniques of using live organisms to produce products and processes useful to humans.
3 EFB (European Federation of Biotechnology) THE INTEGRATION OF NATURAL SCIENCE AND ORGANISMS, CELLS, PARTS THEREOF, AND MOLECULAR ANALOGUES FOR PRODUCTS AND SERVICES
4 PRINCIPLES OF BIOTECHNOLOGY The two core techniques enabled birth of modern biotechnology1. genetic engineering- techniques to alter the chemistry of genetic material (DNA and RNA) to introduce these into host organisms and thus change the phenotype of the host organisms.2. chemical engineering- enable the growth of only desired microbe in large quantities.
5 PRINCIPLES OF BIOTECHNOLOGY Sexual reproduction permits variation.Asexual reproduction preserves the genetic information
6 PRINCIPLES OF BIOTECHNOLOGY Traditional hybridisation very often leads to inclusion and multiplication of undesirable genes along with the desired genes.Recombinant DNA , gene cloning and gene transfer- overcome this limitation and allows us to isolate and introduce only desirable genes.
7 PRINCIPLES AND PROCESSES First instance of creating artificial recombinant DNA1972- Stanley Cohen andHerbert BoyerIsolated DNA encoding for antibiotic resistance from Salmonella typhimuriumand transferred into E. Coli. bacterium
8 PRINCIPLES AND PROCESSES Restriction enzymes-molecular scissorsVectorsLigases-molecular gluesCloning- multiplication of copies of foreignDNA in host cell
9 PRINCIPLES AND PROCESSES Three basic steps in genetically modifying an organism.1.identification of DNA with desirable genes2. introduction of the identified DNA into host3. maintenance of introduced DNA in the host and transfer of the DNA to its progeny
10 PRINCIPLES AND PROCESSES ORIGIN OF REPLICATIONThe foreign DNA should be a part of chromosome having origin of replicationoverview
11 TOOLS OF RECOMBINANT DNA TECHNOLOGY RESTRICTION ENZYMESPOLYMERASE ENZYMESLIGASESVECTORSHOST ORGANISMS
12 TOOLS OF RECOMBINANT DNA TECHNOLOGY RESTRICTION ENZYMESAre enzymes responsible for restricting the growth of bacteriophage (1963)Restriction endonuclease
13 TOOLS OF RECOMBINANT DNA TECHNOLOGY Hind II- the first restriction endonucleaseThey work at specific sequences of bases known as recognition sequencesThere are over 900 restriction enzymes
14 TOOLS OF RECOMBINANT DNA TECHNOLOGY Naming a restriction enzymeEcoRI-First letter- of genera- eg: EscherichiaSecond and third letters- of species- coliFourth letter- strainFifth roman number – the order in which the enzyme isolated
15 TOOLS OF RECOMBINANT DNA TECHNOLOGY NUCLEASESThe class of restriction enzymesExonucleases- remove nucleotidesEndonucleases- cut DNA at specific sites
16 TOOLS OF RECOMBINANT DNA TECHNOLOGY Each restriction endonuclease works at palindromic sequences and make sticky ends -MALAYALAM
17 TOOLS OF RECOMBINANT DNA TECHNOLOGY Working of restriction enzymecut little away from the centre of palindromic sequences leaving sticky endsStickiness is due to hydrogen bonds facilitates action of DNA ligazeIf same restriction enzyme is not used to cut vector and source DNA the recombinant molecule cannot be created
18 TOOLS OF RECOMBINANT DNA TECHNOLOGY Separation and isolation of DNA fragmentsCut DNA fragments can be separated by gel electrophoresisDNA fragments are negatively charged.Separated using a medium – usually agaroseSeparated fragments visualised only after staining the DNA with a compound known as ethidium bromide and exposure to UV
19 TOOLS OF RECOMBINANT DNA TECHNOLOGY ELUTION;the separated bands of DNA are cut out from the agarose gel and extracted from the gel piece.
20 TOOLS OF RECOMBINANT DNA TECHNOLOGY Cloning vectorsPlasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomes DNA.-if an alien piece of DNA is linked with plasmid of bacteriophage we can multiply its numbers Equal to the copy number of the plasmid or bacteriophage.
21 TOOLS OF RECOMBINANT DNA TECHNOLOGY Cloning vectorsFeatures of vectors1. origin of replication- sequence where replication starts-- the selected origin or replication should support high copy number- since it controls the copy number
22 TOOLS OF RECOMBINANT DNA TECHNOLOGY Cloning vectors2. selectable markerHelp in identifying and eliminating non-transformants and permitting the growth of transformants.transformationAre genes encoding resistance to antibiotics such as ampicilin, chloramphenicol etc.
23 TOOLS OF RECOMBINANT DNA TECHNOLOGY Cloning vectors3. cloning sitesCloning sites are recognition sites of restriction enzymesAt antibiotic resistant genepBR322
24 TOOLS OF RECOMBINANT DNA TECHNOLOGY Cloning vectors3. cloning sitesInsertional inactivationα- galactosidase
25 TOOLS OF RECOMBINANT DNA TECHNOLOGY Cloning vectors3. vectors for cloning genes in plants and animalsGene of interest can be introduced into plants or eukaryotic cell through vectors like pathogenic bacteria, virus like Agrobacterioum tumifaciens
26 TOOLS OF RECOMBINANT DNA TECHNOLOGY Competent hostIn order to force bacteria to take up the plasmid, the bacterial cells must first be made competent to take up DNA.MicroinjectionBiolistics or gene gun
27 PROCESSES OF RECOMBINANT DNA TECHNOLOGY ISOLATION OF DNAFRAGMENTATION OF DNAISOLATION OF DERSIRED DNA FRAGMENTLIGATION OF DNA INTO A VECTORTRANSFERING THE RECOMBINANT DNA INTO THE HOSTCULTURING OF HOST CELLS FOR LARGE SCALE EXTRACTION OF PRODUCT
28 PROCESSES OF RECOMBINANT DNA TECHNOLOGY 1. ISOLATION OF THE GENETIC MATERIAL DNADNA SHOULD BE FREE FROM OTHER MACROMOLECULESTreatment with lysozyme (bacteria),cellulase ( plant cells)chitinase (fungus)RNA removed by ribonuclease and proteins by proteaseDNA precipitates after addition of chilled ethanolspooling
29 PROCESSES OF RECOMBINANT DNA TECHNOLOGY Cutting of DNA at specific locationsIncubating purified DNA with restriction enzymesThe joining of DNA is done by mixing gene of interest, cut vector and ligase enzyme
30 PROCESSES OF RECOMBINANT DNA TECHNOLOGY 3. amplification of GENE OF INTEREST using PCRWITH primers and DNA polymerase enzyme.The enzyme is thermostable from Thermus aquaticus
31 PROCESSES OF RECOMBINANT DNA TECHNOLOGY Insertion of recombinant DNA into the host cell/organismThe resistance to antibiotics will act as selectable marker
32 PROCESSES OF RECOMBINANT DNA TECHNOLOGY Obtaining the foreign gene productRecombinant protein- heterologous host
33 PROCESSES OF RECOMBINANT DNA TECHNOLOGY BIOREACTORS\Are used for large scale culture of host cell
34 PROCESSES OF RECOMBINANT DNA TECHNOLOGY DOWN STREAM PROCESSPurification of the biological product