Presentation on theme: "Section H Cloning Vectors"— Presentation transcript:
1 Section H Cloning Vectors Molecular BiologyMolecular BiologySection HCloning Vectors
2 Content . EUKARYOTIC VECTORS . DESIGN OF PLASMID VECTORS Molecular BiologyCloning vectorsContent. DESIGN OF PLASMID VECTORS. BACTERIOPHAGE VECTORS. COSMIDS, YACs AND BACs. EUKARYOTIC VECTORS
3 H1 Design of Plasmid Vectors Molecular BiologyCloning vectorsH1 Design of Plasmid VectorsFig. 1. (a) Screening by insertional inactivation of a resistance gene; (b) replica plating.
4 H1 Design of Plasmid Vectors Molecular BiologyCloning vectorsH1 Design of Plasmid VectorsThe insertion of a DNA fragment interrupts the ORF of lacZ’ gene, resulting in non-functional gene product that can not digest its substrate x-gal.Fig. 2. (a) A plasmid vector designed for blue–white screening; (b) the colonies produced by blue–white screening.
5 H1 Design of Plasmid Vectors Molecular BiologyCloning vectorsH1 Design of Plasmid VectorsAmproripUC18(3 kb)MCS (Multiple cloning sites,多克隆位点）Lac promoterlacZ’Fig. 3. A multiple cloning site at the 5′-end of lacZ′
6 H1-2 A plasmid vector for gene expression Molecular BiologyH1 Design of Plasmid VectorsH1-2 A plasmid vector for gene expressionExpression vectors: allowing the exogenous DNA to be inserted, stored and expressed.Promoter and terminator for RNA transcription are required.Intact ORF and ribosomal binding sites (RBS) are required for translation.
7 (transcription and translation). Molecular BiologyH1 Design of Plasmid VectorsExpression vector(transcription and translation).PromoterslacUV-5: a mutant lac promoter which is independent of cAMP receptor protein.lPL promoterPhage T7 promoterFused proteinsIndividual proteins
8 H1 Design of Plasmid Vectors Molecular BiologyH1 Design of Plasmid VectorsFig. 4. A plasmid designed for expression of a gene using the T7 system
10 λ phage H2 Bacteriophage vector .viruses that can infect bacteria. Molecular BiologyH2 Bacteriophage vectorλ phage.viruses that can infect bacteria..48.5 kb in length.Linear or circular genome (cos ends)Lytic phase (Replicate and release)Lysogenic phase (integrate into host genome)Fig. 1. (a) Phage λ and its genome; (b) the phage λ cos ends.
11 Molecular BiologyH2 Bacteriophage vectorλ replacement vector. Replace the nonessential region of the phage genome with exogenous DNA. high transformation efficiency (1000-time higher than plasmid)
12 H2 Bacteriophage vector Molecular BiologyH2 Bacteriophage vectorProtein coatFig. 2. Cloning in a λ replacement vector.
13 H2 Bacteriophage vector Molecular BiologyH2 Bacteriophage vectorPlaques: the clear areas within the lawn where lysis and re-infection have prevented the cells from growing.Recombinant l DNA may be purified from phage particles from plaques or from liquid culture.
14 H2-2 M13 phage vector H2 Bacteriophage vector Molecular BiologyH2 Bacteriophage vectorH2-2 M13 phage vectorReplication form (RF, dsDNA) of M13 phage can be purified and manipulated like a plamid.Phage particles (ssDNA): DNA can be isolated in a single-stranded form. DNA sequencing.. Site-directed mutagenesis.
16 . Cloning large DNA fragments . Cosmid vectors . YAC vectors Molecular BiologyCloning vectorsH3 COSMIDS, YACs AND BACs. Cloning large DNA fragments. Cosmid vectors. YAC vectors. Selection in S. cerevisiae. BAC vector
17 H3-1 Cloning large DNA fragments Molecular BiologyH3 Cosmids and YACsH3-1 Cloning large DNA fragments(Eukaryotic Genome project)Analysis of eukaryotic genes and genome organization of eukaryotic requires vevtors with a larger capacity for cloned DNA than plasmids or phage λ.
18 H3-2 Cosmid vectors H3 Cosmids and YACs Molecular BiologyH3 Cosmids and YACsH3-2 Cosmid vectorsCosmids use the λ packaging system to package large DNA fragments bounded by λ 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, which are ligated to the ends of target fragments and packaged into λ particles. Cosmids have a capacity for cloned DNA of kb.
19 Formation of a cosmid clone Molecular BiologyFormation of a cosmid cloneFig. 1. Formation of a cosmid clone.
20 H3-3 YAC vectors H3 Cosmids and YACs Molecular BiologyH3 Cosmids and YACsH3-3 YAC vectorsYeast artifical chromosomes can be constructed by ligating the components required for 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 chromosome(YAC) vectors contain two telomeric sequences(TEL), one centromere(CEN), one autonomously replicating sequence(ARS) and genes which can act as selectable markers in yeast.
22 H3-4 Selection in S.cerevisiae Molecular BiologyH3 Cosmids and YACsH3-4 Selection in S.cerevisiaeSelection for the presence of YACs of other vectors in yeast is achived by complementation of a mutant strain unable to produce an essential metabolite, with the correct copy of the mutant gene carried on the vector.
25 H4-1 Yeast episomal plasmids (YEps) Molecular BiologyH4 Eukaryotic VectorsH4-1 Yeast episomal plasmids (YEps)Vectors for the cloning and expression of genes in Saccharomyces cerevisiae.
26 YEp vector H4 Eukaryotic Vectors Replicate as plasmid from 2m origin Molecular BiologyReplicate as plasmid from 2m originH4 Eukaryotic VectorsYEp vectorintegrate by recombinantion
27 H4-2 Agrobacterium tumefaciens Ti plasmid Molecular BiologyH4 Eukaryotic VectorsH4-2 Agrobacterium tumefaciens Ti plasmid
28 Molecular BiologyH4 Eukaryotic Vectorscrown gall or tumor
29 Plant gene engineering using T-DNA vector Molecular BiologyH4 Eukaryotic VectorsPlant gene engineering using T-DNA vector
30 H4-3 Baculovirus H4 Eukaryotic Vectors Molecular BiologyH4 Eukaryotic VectorsH4-3 Baculovirusbaculovirus is an insect virus which is used for the overexpression of animal proteins in insect cell culture.
31 H4-4 Mammalian viral vectors Molecular BiologyH4 Eukaryotic VectorsH4-4 Mammalian viral 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 according to which alternatively spliced transcript is being translated Modified from Fiers et al.,Nature 273:113Fig 2. retrovirus lifecycle
32 Gene transfer H4 Eukaryotic Vectors Molecular BiologyH4 Eukaryotic VectorsGene transferGenes may be introduced into plant of animal cultured cells without the use of a special eukaryotic vector. Bacterial plasnids carrying eukaryotic genes may remain transiently in cells without replication or may integrate into the host genome by recombination at low frequency.