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BIOT E CHNOLO G Y: PRINCIPLES AN D PROC E SSES

BIOT E CHNOLO G Y: PRINCIPLES AN D PROC E SSES. B I O TECH N O L O G Y : P R I NC I PLE S A N D P R OCES S ES. Two core techniques that enabled birth of modern biotechnology:

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BIOT E CHNOLO G Y: PRINCIPLES AN D PROC E SSES

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  1. BIOTECHNOLOGY:PRINCIPLES ANDPROCESSES

  2. BIOTECHNOLOGY:PRINCIPLESANDPROCESSES • Twocoretechniquesthatenabledbirthofmodernbiotechnology: • Geneticengineering:Techniquestoalterthechemistryofgeneticmaterial(DNAandRNA)tointroduceintohostorganismsandthuschangethephenotypeofthehostorganism. • Maintenanceofsterile(microbialcontamination-free)ambientchemicalengineeringprocessestoenablegrowthofonlythedesiredmicrobe/eukaryoticcellinlargequantities

  3. Conceptualdevelopmentoftheprincipleofgeneticengineering • Asexualreproductionpreservesthegeneticidentityofspecies. • Sexualreproductioncreatesvariationandcreatesuniquecombinationsof • geneticmakeup. • Traditionalhybridizationproceduresusedinplantandanimalbreedingleadtoinclusionofundesirablegenesalongwithdesiredgenes. • Thetechniquesofgeneticengineeringwhichincludescreation • ofrecombinantDNA,useofgenecloningandgenetransfer,overcomethislimitationandallowsustoisolateandintroduceonlyoneorasetofdesirablegeneswithoutintroducingundesirablegenesintotargetorganism • Threebasicstepsingeneticallymodifyinganorganism– • IdentificationofDNAwithdesirablegene • IntroductionoftheidentifiedDNAintothehost. • MaintenanceofintroducedDNAinthehostandtransferofthe • DNAtoitsprogeny.

  4. TOOLSOFRECOMBINANTDNATECHNOLOGY • RestrictionEnzymes • Cloningvectors • CompetentHost(fortransformationwithrecombinantDNA)

  5. RestrictionEnzymes • Intheyear1963twoenzymesdiscoveredfromEscherichiacoliwhichrestrictthegrowthofbacteriophageinit. • OneoftheseaddedmethylgroupstoDNA. • OthercutthephageDNA.(restrictionendonuclease) •  • ThefirstrestrictionendonucleasediscoveredisHindII. • HindIIalwayscutDNAmoleculeatparticularpointbyrecognizingaspecificsequenceofsixbasepairs.ThisiscalledrecognitionsequenceforHindII. • Tilldatearound900restrictionenzymesisolatedfrom200strainsofbacteriaeachofwhichrecognizedifferentrecognitionsequences.

  6. Restrictionenzymebelongstonucleases. • Therearetwokindofnucleases: • Exonuclease • Endonuclease  • ExonucleaseremovesnucleotidesfromthefreeendsoftheDNA. • EndonucleasesmakecutsatspecificpositionswithintheDNA. • EachrestrictionendonucleaserecognizesaspecificpalindromicnucleotidesequencesintheDNA. • Palindromesarethegroupoflettersthatreadsamebothforwardand backward, e.g. “MALAYALAM”. • ThepalindromeinDNAisasequenceofbasepairsthatreadssameon thetwostrandswhenorientationofreadingiskeptsame. 

  7. TherestrictionenzymecutthestrandofDNAlittleawayfromthecentreofthepalindromesites,butbetweenthesametwobasesontheoppositestrand.Thisleavessinglestrandedportionsattheends.ThereareoverhangingstretchescalledstickyTherestrictionenzymecutthestrandofDNAlittleawayfromthecentreofthepalindromesites,butbetweenthesametwobasesontheoppositestrand.Thisleavessinglestrandedportionsattheends.Thereareoverhangingstretchescalledsticky • endsoneachstrand. • ThisstickinessoftheendsfacilitatestheactionoftheenzymeDNAligases. • TheforeignDNAandthehostDNAcutbythesamerestrictionendonuclease,theresultantDNAfragmentshavethe same kind of „sticky-ends‟ and these can be joined together usingDNAligases.

  8. Conventionfornamingrestrictionendonuclease • Thefirstletterofthenamecomesfromthegenus. • Secondtwoletterscomefromthespeciesoftheprokaryoticcellfromwhichtheenzymeisolated • ThefourthletterisincapitalformderivedfromtheStrainof • microbes. • TheRomanletterfollowedistheorderofdiscovery • Bestexample:EcoRIcomesfromEscherichiacoliRY13

  9. SeparationandisolationofDNAfragments • ThecuttingofDNAbyrestrictionendonucleasesresultsinthefragmentsofDNA. • Thesefragmentsareseparatedbyatechnique • calledgelelectrophoresis. • SincetheDNAfragmentsarenegativelycharged,theycanbeseparatedbyforcingthemtomovetowardsanodeunderanelectricfieldthroughamedium/matrix. • Mostcommonlyusedmatrixisagarose,anatural • polymerextractedfromseaweed. • DNAfragmentsseparateaccordingtotheirsizethroughsievingeffectprovidedbytheagarosegel.Hencethesmallerthefragmentsize,fartheritmoves. • TheseparatedfragmentsarevisualizedbystainingthemwithEthidiumbromidefollowedbyexposuretoUVradiation. • TheseparatedbandsofDNAarecutoutfromtheagarosegelandextractedfromthegelpiece.Thisstepiscalledelution.

  10. Cloningvectors: • TheplasmidandbacteriophageshavetheabilitytoreplicatewithinbacterialcellsindependentofthecontrolofchromosomalDNA. • AlienDNAlinkedwiththevectormultiplyitsnumberequaltothecopynumberoftheplasmidorbacteriophage. • Features of cloning vector:Originofreplication: • Thisisthesequencewherethereplicationstartscalledorigene. • ThealienDNAlinkedwithvectoralso • replicates. • ControlsthecopynumberofthelinkedDNA.

  11. Selectablemarker • Itisrequiredtoidentifyrecombinantfromthenon-recombinant. • Helpsinidentifyingandeliminatingnon-transformantsand • selectivelypermittingthegrowthofthetransformants. • TransformationisaprocedurethroughwhichapieceofforeignDNAisintroducedinahostbacterium. • Normally,thegenecodingresistancetoantibioticssuchasampicilin.Tetracycline,chloramphenicolorkanamycinsetcareconsideredasusefulselectablemarkersforE.coli. • ThrnormalE.colicellsdonotcarryresistanceagainstanyof • antibiotics.

  12. Cloningsites • InordertolinkthealienDNA,thevectorneedstohaveveryfew,preferablysingle,recognitionsites(palindromicsite)forthecommonlyusedrestrictionendonuclease. • CommonlyusedvectorispBR322,forE.coli. • TheligationofforeignDNAiscarriedoutatarestrictionsitepresentinoneofthetwoantibioticresistancegenes. • IfaforeignDNAligatedorinsertedattheBamHIsiteoftetracyclineresistancegeneinthevectorpBR322,therecombinantplasmidwilllosetetracyclineresistance.(insertionalinactivation) •  • Therecombinantcanbeidentifiedfromthenon-recombinantinfollowingsteps: • Allaregrowninampicilinmedium • Onereplicaofaboveplategrowninampicilinmedium(control) • Otherreplicagrowninthemediumcontainingbothtetracyclineandampicilin. • Thecoloniesgrowsinplate-Ibutfailedtogrowinplate-IIareidentifiedasrecombinants

  13. Alternativeselectablemarker • InE.coliaplasmidcalledPUK-18isusedasselectablemarker,which • isbetterthanpBR322. • TheforeignDNAisintroducedwithinthecodingsequenceofanenzyme β-galactosidase,whichconvertX-Gal(chromatogenicsubstrate)intoGalactoseand5-bromo+4chloroindigo(bluecolor) • Thenon-recombinantproduceenzymeandgivebluecoloredcolonies. • Therecombinantunabletoproduceβ-galactosidaseanddoesnotproducebluecoloredcoloniesafteradditionofchromatogenicsubstratei.e.X-Gal. • ThisinactivationofinsertionofforeignDNAcalledinsertional • inactivation

  14. Vectorsforcloninggenesinplantsandanimals • Agrobacteriumtumefaciens,apathogenicbacteriumofseveraldicotplants. • ThisbacteriumcontainsaplasmidcalledTi-plasmid.(tumor inducing) • InnaturalconditiontheA.tumifacienstransfertheT-DNAintotheplantwhichtransformnormalplantcellsintoatumoranddirectthesetumorcellstoproducethechemicalrequiredbythepathogen. • Retrovirusesinanimalshavetheabilitytotransformnormalcells intocancerouscells. • Thedis-armedretrovirusesarebeingusedtotransfergeneintoanimals. • InTi-plasmidtheT-DNAisreplacedbythegeneofinterest,stillA.tumifaciensabletotransferthegeneintotheplantwithoutcausingtumorinplants.

  15. CompetentHost(fortransformationwithrecombinant DNA) • DNAisahydrophilicmolecule;itcannotpassthroughcellmembranes. • Inordertoforcebacteriatotake-uptheplasmid,thebacterialcells • must first be made„competent‟ to take up DNA. • Thebacterialcellistreatedwithdivalentcationssuchascalcium,whichincreasestheefficiencyofDNAuptakebythebacteria. • RecombinantDNAandthebacterialcellsareincubatedinice,followedbyplacingthembrieflyat42oC(heatshock)andthenputtingthembackinice. • BymicroinjectiontherecombinantDNAdirectlyinjectedintothenucleusoftheanimalcell. • Plantcellsarebombardedwithhighvelocitymicro-particlesofgoldortungstencoatedwithDNAinamethodknownasbiolisticsorgenegun. • ThedisarmedpathogenvectorswhichwhenallowedinfectingthecelltransfertherecombinantDNAintothehost.

  16. PROCESSOFRECOMBINANTTECHNOLOGY: • IsolationofDNA, • FragmentationofDNAbyrestrictionendonuclease. • IsolationofdesiredDNAfragmentbygelelectrophoresis. • LigationofDNAfragmentwithavectorbyDNAligase • TransferringtherecombinantDNAintothehost • Culturingthehostcellsinamediumatlargescalein • abioreactor. • Extractionofdesiredproductbydownstreamprocessing.

  17. IsolationoftheGeneticmaterial(DNA): • BacterialcellwalldigestedbyLysozyme. • Plantcellwallisdigestedbycellulaseandpectinase. • Fungalcellwallisdigestedbychitinase. • RNAofthecellularcontentisdigestedbyribonuclease. • ProteinsareremovedbyProteases. • PurifiedDNAultimatelyprecipitatedoutafteraddition • ofchilledethanol. • TheprecipitatedDNAisseparatedandremoved • byspooling.

  18. AmplificationofGeneofInterestusingPCR • PCRstandsforPolymerasechainreaction: • Multiplecopyofgeneofinterestcanbesynthesizedinvitro. • PCRincludesfollowingsteps: • Denaturation • DoublestrandedDNAmadesinglestranded. • ItisdonebyheatingtheDNAat94oC. • EachsinglestrandedDNAiscalledTemplatestrand

  19. Annealing • Twosetsofprimer(smalloligonucleotidechainthatarecomplementary to the DNA at 3‟ end of the DNA template) addedtothemedium. • Thisisdoneataround50oC. • Extension • Deoxyribonucleotidestriphosphatesareaddedinthemedium. • Taqpolymerasecatalysesthepolymerizationreactionusingnucleotides extending from the primer towards 5‟ end of the template. • Taqpolymeraseisathermostablepolymeraseisolatedfroma • bacteriumcalledThermusaquaticus. • Itcatalysespolymerizationreactionat74oC.

  20. ObtainingtheForeignGeneproductorRecombinantproduct: • Theproteinencodinggeneisexpressedin • aheterogeneoushostiscalledarecombinantprotein. • Thehostisculturedinacontinuousculturesystemprovided • inbioreactor. • Abioreactorprovidesoptimumgrowthconditions(temperature,pH,substrate,salts,vitamins,oxygen) • Bioreactorcoverttherawmaterialsintospecificproduct, • specificenzyme.

  21. Downstreamprocessing • Afterbiosynthesisinsidethebioreactor,theproducthastobesubjectedthroughaseriesofprocessesbeforeitisreadyformarketing. • Theprocessincludesseparationandpurification,whicharecollectivelyreferredasdownstreamprocessing. • Theproducthastobeformulatedinsuitablepreservatives. • Suchformulationhastoundergothroughclinicaltrialsasincaseofdrugs.

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