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
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