Section H 克隆载体.ppt

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1、Section H Cloning VectorsMolecular BiologyMolecular BiologyMolecular BiologyCharacters of cloning vectorsCloning vectorsMolecular BiologyMolecular BiologyCloning vectors.DESIGN OF PLASMID VECTORS.DESIGN OF PLASMID VECTORS.BACTERIOPHAGE VECTORS.BACTERIOPHAGE VECTORS.COSMIDS,YACs AND BACs.COSMIDS,YACs

2、 AND BACs.EUKARYOTIC VECTORS EUKARYOTIC VECTORS ContentMolecular BiologyMolecular Biology天然存在的野生型质粒由于分子量天然存在的野生型质粒由于分子量大、拷贝数低、单一酶切位点少、大、拷贝数低、单一酶切位点少、遗传标记不理想等缺陷，不能满足遗传标记不理想等缺陷，不能满足克隆载体的要求，因此往往需要以克隆载体的要求，因此往往需要以多种野生型质粒为基础进行人工构多种野生型质粒为基础进行人工构建。目前已开发出更有效的、便于建。目前已开发出更有效的、便于筛选的一系列载体。筛选的一系列载体。H1 Design of

3、Plasmid VectorsH1 Design of Plasmid VectorsCloning vectorsMolecular BiologyMolecular BiologyTwin antibiotic resistanceH1 Design of Plasmid VectorsMolecular BiologyMolecular BiologyH1 Design of Plasmid VectorsMolecular BiologyMolecular BiologyH1 Design of Plasmid VectorsMolecular BiologyMolecular Bio

4、logyH1 Design of Plasmid VectorsMolecular BiologyMolecular BiologyBlue-white screeningH1 Design of Plasmid VectorsMolecular BiologyMolecular BiologyThe insertion of a DNA fragment interrupts the ORF of lacZ gene,resulting The insertion of a DNA fragment interrupts the ORF of lacZ gene,resulting in n

5、on-functional gene product that can not digest its substrate x-gal.in non-functional gene product that can not digest its substrate x-gal.H1 Design of Plasmid VectorsMolecular BiologyMolecular BiologySo How Do You Know IfYou Cloned Something?IPTG-Induces expression of lacZX-Gal-A lactose analog whic

6、h turns blue when split by b-galactosidaseAmpicillin-Kills all bacteria that lack the plasmidCloning vectorsMolecular BiologyMolecular BiologyOOHColonies containing vector WITHOUT an insert are blue.+ampicillin+X-gal(We dont want these.)BamH1oriApRlacZMolecular BiologyMolecular BiologyH1 Design of P

7、lasmid VectorsWhen foreign DNA is inserted,it inactivates lacZnbeta-galactosidase is not madenX-gal is not cleaved ncolonies with insert are white,NOT blueXX-gal(CLEAR)+ampicillin+X-galX(LacZ-)insertMolecular BiologyMolecular BiologyH1 Design of Plasmid VectorsMolecular BiologyMolecular BiologyH1 De

8、sign of Plasmid VectorsFig.3.A multiple cloning site at the 5-end of lacZ AmpAmpr roripUC18(3 kb)MCS(Multiple cloning sites,多克隆位点）Lac promoterlacZMolecular BiologyMolecular BiologyH1 Design of Plasmid VectorsMultiple cloning sites,多克隆位点多克隆位点H1-2 A plasmid vector for gene expressionExpression vectors

9、:Expression vectors:allowing the exogenous DNA to be inserted,stored and allowing the exogenous DNA to be inserted,stored and expressed.expressed.1.1.PromoterPromoter and and terminatorterminator for RNA transcription are required.for RNA transcription are required.2.2.Intact ORFIntact ORF and and r

10、ibosomal binding sites(RBS)ribosomal binding sites(RBS)are required for are required for translation.translation.H1 Design of Plasmid VectorsMolecular BiologyMolecular BiologyExpression vector Expression vector(transcription and translation).(transcription and translation).PromotersPromoters1.1.lacU

11、V-5lacUV-5:a mutant lac promoter which is independent of:a mutant lac promoter which is independent of cAMP receptor protein.cAMP receptor protein.2.2.lP lPL L promoter promoter3.3.Phage T7 promoterPhage T7 promoter Fused proteinsFused proteinsIndividualIndividual proteinsH1 Design of Plasmid Vector

12、sMolecular BiologyMolecular BiologyH1 Design of Plasmid VectorsMolecular BiologyMolecular BiologyFig.4.A plasmid designed for expression of a gene using the T7 system An Expression-Cloning VectorAn Expression-Cloning VectorH1 Design of Plasmid VectorsMolecular BiologyMolecular BiologyFactors affecti

13、ng protein expression Gene copy number Promoter strength®ulation Translation initiation Codon usage Protein and mRNA stabilityH1 Design of Plasmid VectorsMolecular BiologyMolecular BiologyH2 Bacteriophage vector Tow examples:Tow examples:H2-1 H2-1 phagephage bacteriophage bacteriophage replacemen

14、t vector replacement vector H2-2 H2-2 M13 phageM13 phage M13 phage vectorM13 phage vector Cloning in M13 Cloning in M13 Hybrid plasmid-M13 vectors Hybrid plasmid-M13 vectorsCloning vectorsMolecular BiologyMolecular Biology.viruses that can infect bacteria.viruses that can infect bacteria.48.5 kb in

15、length.48.5 kb in length.Linear or circular genome(cos ends).Linear or circular genome(cos ends)phagephageLytic phase Lytic phase(Replicate and release)(Replicate and release)Lysogenic phase Lysogenic phase(integrate into host genome)(integrate into host genome)H2 Bacteriophage vectorFig.1.(a)Phage

16、and its genome;(b)the phage cos ends.Molecular BiologyMolecular Biology replacement vector.Replace the nonessential region of the phage genome.Replace the nonessential region of the phage genome with exogenous DNAwith exogenous DNA.high transformation efficiency(1000-time higher.high transformation

17、efficiency(1000-time higher than plasmid)than plasmid)H2 Bacteriophage vectorMolecular BiologyMolecular BiologyProtein coatH2 Bacteriophage vectorFig.2.Cloning in a replacement vector.Molecular BiologyMolecular BiologyPlaques:Plaques:the clear areas within the lawn where lysis and re-infection have

18、the clear areas within the lawn where lysis and re-infection have prevented the cells from growing.prevented the cells from growing.Recombinant l DNARecombinant l DNA may be purified from phage particles from plaques or from may be purified from phage particles from plaques or from liquid culture.li

19、quid culture.H2 Bacteriophage vectorMolecular BiologyMolecular BiologyH2-2 M13 phage vectorH2 Bacteriophage vector1.1.Replication form(RF,dsDNA)Replication form(RF,dsDNA)of M13 phage can be purified and of M13 phage can be purified and manipulated like a plamid.manipulated like a plamid.2.2.Phage pa

20、rticles(ssDNA):Phage particles(ssDNA):DNA can be isolated in a single-stranded formDNA can be isolated in a single-stranded form.DNA sequencing.DNA sequencing.Site-directed mutagenesis.Site-directed mutagenesis.Molecular BiologyMolecular BiologyM13 mp18 vector Molecular BiologyMolecular BiologyH3 CO

21、SMIDS,YACs AND BACs.Cloning large DNA fragments.Cloning large DNA fragments.Cosmid vectors.Cosmid vectors.YAC vectors.YAC vectors.Selection in S.cerevisiae.Selection in S.cerevisiae.BAC vector.BAC vectorCloning vectorsMolecular BiologyMolecular Biology Analysis of eukaryotic genes and genome Analysi

22、s of eukaryotic genes and genome organization of eukaryotic requires vevtors with a organization of eukaryotic requires vevtors with a larger capacity for cloned DNA than plasmids or larger capacity for cloned DNA than plasmids or phage phage.H3-1 Cloning large DNA fragments(Eukaryotic Genome projec

23、t)(Eukaryotic Genome project)H3 Cosmids and YACsMolecular BiologyMolecular BiologyH3-2 Cosmid vectors Cosmids use the Cosmids use the packaging system to package large DNA fragments bounded by packaging system to package large DNA fragments bounded by cos sites,which circularize and replicate as pla

24、smids after infection of E.coli cells.cos sites,which circularize and replicate as plasmids after infection of E.coli cells.Some cosmid vectors have two cos sites,and are cleaved to produce two cos ends,Some cosmid vectors have two cos sites,and are cleaved to produce two cos ends,which are ligated

25、to the ends of target fragments and packaged into which are ligated to the ends of target fragments and packaged into particles.particles.Cosmids have a capacity for cloned DNA of 30-45 kb.Cosmids have a capacity for cloned DNA of 30-45 kb.H3 Cosmids and YACsMolecular BiologyMolecular BiologyFormati

26、on of a cosmid cloneFig.1.Formation of a cosmid clone.Molecular BiologyMolecular BiologyYeast artifical chromosomes can be constructed by ligating the components required for Yeast artifical chromosomes can be constructed by ligating the components required for replication and segreation of natural

27、yeast chromosomes to very large fragments of replication and segreation of natural yeast chromosomes to very large fragments of target DNA,which may be more than 1 Mb in length.Yeast artifical target DNA,which may be more than 1 Mb in length.Yeast artifical chromosome(YAC)vectors contain two telomer

28、ic sequences(TEL),one chromosome(YAC)vectors contain two telomeric sequences(TEL),one centromere(CEN),one autonomously replicating sequence(ARS)and genes which can centromere(CEN),one autonomously replicating sequence(ARS)and genes which can act as selectable markers in yeast.act as selectable marke

29、rs in yeast.H3-3 YAC vectorsH3 Cosmids and YACsMolecular BiologyMolecular BiologyH3 Cosmids and YACsMolecular BiologyMolecular Biology Selection for the presence of YACs of other vectors in yeast Selection for the presence of YACs of other vectors in yeast is achived by complementation of a mutant s

30、train unable to is achived by complementation of a mutant strain unable to produce an essential metabolite,with the correct copy of the produce an essential metabolite,with the correct copy of the mutant gene carried on the vector.mutant gene carried on the vector.H3-4 Selection in S.cerevisiaeH3 Co

31、smids and YACsMolecular BiologyMolecular BiologyH4 Eukaryotic Vectors1.Shuttle vectors1.Shuttle vectors2.Yeast episomal plasmids(Yeasts)2.Yeast episomal plasmids(Yeasts)3.Agrobacterium tumefaciens Ti plasmid(Plants)3.Agrobacterium tumefaciens Ti plasmid(Plants)4.Baculovirus(Insects)4.Baculovirus(Ins

32、ects)5.Mammalian viral vectors(Mammalian)5.Mammalian viral vectors(Mammalian)Cloning vectorsMolecular BiologyMolecular BiologyShuttle vectorsH4 Eukaryotic VectorsMolecular BiologyMolecular Biology H4-1 Yeast episomal plasmids(YEps)H4 Eukaryotic VectorsVectors for the cloning and expression of genes

33、in Vectors for the cloning and expression of genes in Saccharomyces cerevisiaeSaccharomyces cerevisiae.Molecular BiologyMolecular BiologyReplicate as plasmid Replicate as plasmid from 2m originfrom 2m originintegrate by integrate by recombinantionrecombinantionYEp vectorYEp vectorH4 Eukaryotic Vecto

34、rsMolecular BiologyMolecular BiologyH4-2 Agrobacterium tumefaciens Ti plasmidH4-2 Agrobacterium tumefaciens Ti plasmidH4 Eukaryotic VectorsMolecular BiologyMolecular Biologycrown gall or tumorcrown gall or tumorH4 Eukaryotic VectorsMolecular BiologyMolecular BiologyPlant gene engineering Plant gene

35、engineering using T-DNA vectorusing T-DNA vectorH4 Eukaryotic VectorsMolecular BiologyMolecular BiologyH4-3 BaculovirusH4 Eukaryotic Vectorsbaculovirus is an insect virus which is used for the overexpression of animal proteins in insect cell culture.Molecular BiologyMolecular BiologyH4-4 Mammalian v

36、iral vectorsH4 Eukaryotic VectorsFig 1.Gene expression by SV40.Early genes are in red,late genes are in green.Note:-indicates regions of the primary transcript which are removed in the alternatively processed mRNA.Cross-hatched area indicates region of RNA translated in different reading frames acco

37、rding to which alternatively spliced transcript is being translated Modified from Fiers et al.,Nature 273:113 Fig 2.retrovirus lifecycleMolecular BiologyMolecular BiologyGene transferH4 Eukaryotic VectorsGenes may be introduced into plant of animal cultured cells Genes may be introduced into plant o

38、f animal cultured cells without the use of a special eukaryotic vector.Bacterial plasnids without the use of a special eukaryotic vector.Bacterial plasnids carrying eukaryotic genes may remain transiently in cells without carrying eukaryotic genes may remain transiently in cells without replication or may integrate into the host genome by replication or may integrate into the host genome by recombination at low frequency.recombination at low frequency.Molecular BiologyMolecular Biology