Vectors in biotechnolgy

Vectors in biotechnolgy

1. VECTORS IN
BIOTECHNOLGY
Presented By-BaisaliDora
SonaliPati
+3 1
st
Yr. –Botany Hons.
Under The Guidance of
Faculties of Dept. of Botany,
V. Dev (Auto.) College, Jeypore
Dated-23/ Feb/2015

2. DEFINATION
ThesearecarrierorvehicularDNAmolecules,
whichcarrygeneofinterest.Avectorwhen
combinedwiththegeneofinterest;arecombinant
DNAmoleculeisobtained.
TheaimofusingvectorinRDTisoneofthe
following
Toobtainmultiplecopiesofthegeneofinterest.Inthis
caseweusecloningvectorsor
Toobtaintheproductofgeneofinterest.Inthiscase
weuseexpressionvectors
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3. CHARECTERISTIC FEATURES OFANIDEAL
VECTORS
Itshouldbeabletoreplicateautonomously.
Itshouldbeeasytoisolateandpurify.
Transformationofhostwiththevectorshouldbe
easy.
Itshouldhavesuitablemarkergenesthatallow
easydetection.
Itshouldhavetheabilitytointegrateeitheritselfor
theDNAinsertitcariesintothegenomeofhostcell.
Itshouldcontainsuniquetargetsites.
Itshouldcontainatleastsuitablecontrolelements,
e.g.promoter,operator,andribosomebindingsites
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4. GENERALCONSTRUCTION OFVECTORS
ADNAmoleculeshouldpossesthefollowingtwo
essentialcharacteristicstoactascloningvector.
Originofreplication-itisrequiredforautonomous
replicationofplasmidusingthehostreplication
machinary
Selectablemarkers-4types
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5. Selectablemarkers-
Antibioticsresistancemarkergenes
Herbicidestolerantmarkergenes(Hb
r
)
Metabolic/auxotrophicmarkergenes
Screenablemarkergenes
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6. CLASSIFICATIONOFVECTORS
Onthebasisofouraimwithgeneofinterest;-
1.cloningvectors
2.expressionvectors
Onthebasisofhostcellused;-
1.vectorsforbacteria
2.vectorsforyeast
3.vectorsforanimals
4.vectorsforplants
oOnthebasisofcellularnatureofvectorsofhostcell
1.Prokaryoticvectors
2.Eukaryoticvectors 6
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7. Plasmidbasedandbacteriophagevectorsaremost
commonprokaryoticvectors
Theprokaryoticvectorsinclude:-plasmidderived
vectors,bacteriophagederivedvectors,phagemid
vectors,plasmidvectorsandfosmidvectors
Plasmidvectors
Thesearethemostcommonvectorsforthe
prokaryotichostcell.
Plasmidsaresmall,circular,doublestrandedDNA
moleculeslackingproteincoatexistnaturallyinthe
cytoplasmofmanystrains.
Ex.pBR322,pUCvector
Prokaryotic vectors
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8. PBR322:
Thiswasthefirstwidelyusedplasmidvector.
pBR322hasarelativelysmallsizeof4.363bp.
Alsothisvectorhasareasonablyhighcopynumber
(~15copiespercell).
NomenclatureofpBR322:
p-Plasmid
BR-BoliverandRodriguez-
Thetworesearcherswhodevelopedit.
322-No.ofdevelopedinthesamelaboratory.
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9. ConstructionofpBR322:
1.Originofreplication
2.Selectablemarkers–Itcarries2antibioticresistancegenes
(AmpicillinandTetracycline)
3.Cloningsites
UsesofpBR322:
Itiswidelyusedascloningvector.
Itiswidelyusedasamodelsystemforstudyofprokaryotic
transcriptionandtranslation.
AdvantagesofpBR322:
Duetoitssmallsize(~4.4kb)enableseasypurificationand
manipulation.
2selectablemarkers(AmpandTet.)alloweasyselectionof
recombinantDNA.
Itcanbeamplifiedupto1000-3000copies.
DisadvantagesofpBR322:
Ithasveryhighmobility.
Thereisalimitationinthesizeofgenesofinterestthatitcan
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10. II. PUC VECTORS:
pUCareobtainedbymodifyingthepBR322vectors.
ThesearesmallerthanpBR322ofbeingonly~2.7kb.
Itproduce500-600copies.
NomenclatureofpUCvectors:
P-plasmid
UC-UniversityofCalifornia
ConstructionofpUC:
1.Originofreplication
2.Selectablemarkers
3.Lac-zgenehavingMCS(MultipleCloningSites)
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11. UsesofpUCvectors:
pUCvectorscanbeusedbothascloningvector
andexpressionvector.
AdvantagesofpUCvectors:
1.Produceshighcopynumberof500-600copies
percell.
2.Easyandsinglestepselection.
DisadvantagesofpUCvector:
Itcannotaccommodateageneofinterestlarger
than15kb.
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12. III. FOSMIDVECTORS
Thesearesimilartocosmidvectors,
butarebasedonbacterialF-Plasmids.
Fosmidsare40kbofrandomgenomicDNA.
IV. BACTERIOPHAGE DERIVEDVECTORS
Bacteriophages/phagesarecommonlyknownvirusesthatinfect
bacteria.
Likeviruses,phagesareverysimpleinstructuresconsistingmerelyof
DNA.
Ex.Lambda(λ)phagesvectors,Bacteriophagesm13vectors
Lambda(λ)phagesvectors.
Thesearewidelyusedvectorsforcloningofverylargepiecesof
genes.
Lambdaisatypicalexampleofheadandtailphage.Thelambda
moleculesis49kbinsize. 12
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13. Typesoflambda(λ)vectors:
1.Lambdainsertionvectors
2.Lambdareplacementvector
Advantagesoflambda(λ)vectors:
I.Storageofphageparticlesiscomparativelymucheasier
thanthatofplasmidbasedvector.
II.Theself-lifeofphageparticlesisinfinite.
III.TransfectionofE.coliismucheasierwithphage
particles.
Disadvantagesoflambda(λ)vectors:
I.Itisdifficulttoisolate.
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14. V. COSMIDVECTORS
ItisthemostsophisticatedtypeofLambdabasedvector.Cosmidare
hybridbetweenphageDNAmoleculeandvectorplasmid.
ConstructionsofCosmidVectros:
Cosmidisbasicallyaplasmidthatcarriesacossite.
ItcontainselectablemarkerssuchasAmpicillinresistant(Amp
r
)gene
andaoriginofreplication.
Cosmidlacksallthelambdagenes,soitdoesn’tproduceplaques.
UsesofCosmidVectros:
Usedforconstructionofgenomiclibrariesofeukaryotes.
AdvantagesofCosmidVectros:
Usedtoclonegeneofinterestupto40kb.
Easyscreeningmethodisfound
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15. VI. PHAGEMIDVECTORS
Phagemid(phagefromM13bacteriophageandmidfromplasmid).
Itcontains1500bp.
ConstructionsofPhagemid:
1.Originofreplication
2.Lac-Zgene
3.Multiplecloningsites
4.Originofreplication
5.Amp
r
resistantgene
UsesofPhagemid:
Usedascloningvectors,sequenceinvectors,expressionvectors
AdvantagesofPhagemid:
Itcanbeusedtoprovidesingleordoublestrandedmaterialwithout
anyrecloning.
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16. VI. PHASMIDVECTOR
Thesearetrulyplasmidwithphagegenes.
ThesearelinearduplexDNAwhoseendsare
lambdasegmentsthatcontainsallthegenes.
Boththelambdaandplasmidreplicationfunction
areintact.
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17. Yeast,animalandplantvectorsareallconsidered
aseukaryoticvectors.
Yeastvectors:
Yeastplaysveryimportantroleinbrewingand
breadmaking.
Itcanbeusedinpharmaceuticalsfromcloned
genes.
EUKARYOTIC VECTORS
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18. GeneralconstructionYeastVectors:
1.Allofthemcontainsuniquetargetsitesforno.of
restrictionendonucleases.
2.Highcopynumber.
3.Employmarkers.
TypesofYeastVectors:
Itcanbedividedinto3types:
a.Yeastcloningvector.
b.Yeastexpressionvectors.
c.Yeastartificialchromosomes(YAC)
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19. ANIMAL VECTORS
1.BaculovirusVectors:
Itinfectsinsects.
Thisvirusisrodshapedwithalargedouble
strandedgenome.
ThegeneofInterestisexpressedduringthe
infectionandveryhighyieldsofprotein,canbe
achievedbythetimelysis.
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20. 2.BovinePapillomaVirusVector:
BPVcauseswartsincattle.
BPVhasacapsidproteinsurroundingacircular
doublestrandedDNAofsize79kb.
69%ofthisgenomeisimp.forviralfunction,31%
ofgenomecanbereplacedbyanyforeignDNA
sequence.
Ex-p
3
.7LDL
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21. 3.SV40VirusBasedVector:
Itisasphericalviruscontainsdoublestranded
circularDNAofsize5.2kb.
Theviralproteincontains3viralcodedproteins;out
ofwhichlargeT-Proteinisimp.forviralDNA
replication.
TypesofSV40:
SV40PassiveTransformingVector
SV40TransducingVectors
SV40PlasmidVectors
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22. PLANTVECTORS
ThesevectorsareusedtoproduceGenetically
Modified(GM)crops.
ShuttleVectors:
CanMulti[plyinto2differentunrelatedspecies.
Contains2orisitesforreplication
TypesofShuttleVectors:
Eukaryotic-ProkaryoticShuttlevectors
Prokaryotic-ProkaryoticShuttlevectors
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23. ARTIFICIALCHROMOSOMES
SyntheticallydesignedDNAmoleculesofknown
structures;whichareassembledinvitrofrom
specificDNAsequencesthatactslikeanatural
chromosomes.
Mainlycontain3components-
Centromere
2telomeres
Origin(Ori)ofReplication
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24. TypesofArtificialchromosomes:
1.YeastArtificialChromosomes(YAC):
Uses:
HelpinproductionGeneLibraries
Canbeusedtostudyvariousaspectsofchromosome
structureandbehaviorduringMeiosis.
2.BacterialArtificialChromosomes(BAC):
CloningVectorsbasedonFfactorofE.coliplasmid.
Uses:
HelpinstudyingNeurologicaldiseaseviz.Alzheimer’s
DiseaseinMice
IncaseofAneuploidy,itisassociatedwithDown’sSyndrome.
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25. 3.HumanArtificialChromosomes(HAC):
47artificialminichromosome
Uses:
UsedinExpressionStudiesasGeneTransfer
Vector
4.P1DerivedArtificialChromosomes(PAC):
DerivedfromDNAofP1bacteriophage
Uses:
UsedtocloneDNAfragmentsofE.colicells 25
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