多克隆位点).ppt

Section H Cloning Vectors * * Molecular Biology Molecular Biology Cloning vectors . DESIGN OF PLASMID VECTORS . BACTERIOPHAGE VECTORS . COSMIDS, YACs AND BACs . EUKARYOTIC VECTORS Content Molecular Biology H1 Design of Plasmid Vectors Cloning vectors Fig. 1. (a) Screening by insertional inactivation of a resistance gene; (b) replica plating. Molecular Biology H1 Design of Plasmid Vectors Cloning vectors Fig. 2. (a) A plasmid vector designed for blue–white screening; (b) the colonies produced by blue–white screening. The 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. Molecular Biology H1 Design of Plasmid Vectors Cloning vectors Fig. 3. A multiple cloning site at the 5′-end of lacZ′ Ampr ori pUC18 (3 kb) MCS (Multiple cloning sites, 多克隆位点） Lac promoter lacZ’ Molecular Biology H1-2 A plasmid vector for gene expression Expression 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. H1 Design of Plasmid Vectors Molecular Biology Expression vector (transcription and translation). Promoters lacUV-5: a mutant lac promoter which is independent of cAMP receptor protein. lPL promoter Phage T7 promoter Fused proteins Individual proteins H1 Design of Plasmid Vectors Molecular Biology H1 Design of Plasmid Vectors Fig. 4. A plasmid designed for expression of a gene using the T7 system Molecular Biology H2 Bacteriophage vector Tow examples: H2-1 λ phage bacteriophageλ λ replacement vector H2-2 M13 phage M13 phage vector Cloning in M13 Hybrid plasmid-M13 vectors Cloning vectors Molecular Biology .viruses that can infect bacteria. .48.5 kb in length .Linear or circular genome (cos ends) λ phage Lytic phase (Replicate and release) Lysogeni