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Section H Cloning Vectors Molecular Biology Cloning vectors. DESIGN OF PLASMID VECTORS. BACTERIOPHAGE VECTORS. COSMIDS, YACs AND BACs. EUKARYOTIC VECTORS.

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Presentation on theme: "Section H Cloning Vectors Molecular Biology Cloning vectors. DESIGN OF PLASMID VECTORS. BACTERIOPHAGE VECTORS. COSMIDS, YACs AND BACs. EUKARYOTIC VECTORS."— Presentation transcript:

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2 Section H Cloning Vectors Molecular Biology

3 Cloning vectors. DESIGN OF PLASMID VECTORS. BACTERIOPHAGE VECTORS. COSMIDS, YACs AND BACs. EUKARYOTIC VECTORS Content Molecular Biology

4 H1 Design of Plasmid Vectors Cloning vectors Fig. 1. (a) Screening by insertional inactivation of a resistance gene; (b) replica plating. Molecular Biology

5 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

6 H1 Design of Plasmid Vectors Cloning vectors Fig. 3. A multiple cloning site at the 5′-end of lacZ′ Amp r ori pUC18 (3 kb) MCS (Multiple cloning sites, 多克隆位点) Lac promoter lacZ’ Molecular Biology

7 H1-2 A plasmid vector for gene expression Expression vectors: allowing the exogenous DNA to be inserted, stored and expressed. 1.Promoter and terminator for RNA transcription are required. 2.Intact ORF and ribosomal binding sites (RBS) are required for translation. H1 Design of Plasmid Vectors Molecular Biology

8 Expression vector (transcription and translation). Promoters 1.lacUV-5: a mutant lac promoter which is independent of cAMP receptor protein. 2.lP L promoter 3.Phage T7 promoter Fused proteins Individual proteins H1 Design of Plasmid Vectors Molecular Biology

9 H1 Design of Plasmid Vectors Fig. 4. A plasmid designed for expression of a gene using the T7 system Molecular Biology

10 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

11 .viruses that can infect bacteria kb in length.Linear or circular genome (cos ends) λ phage Lytic phase (Replicate and release) Lysogenic phase (integrate into host genome) H2 Bacteriophage vector Fig. 1. (a) Phage λ and its genome; (b) the phage λ cos ends. Molecular Biology

12 λ replacement vector. Replace the nonessential region of the phage genome with exogenous DNA. high transformation efficiency (1000-time higher than plasmid) H2 Bacteriophage vector Molecular Biology

13 Protein coat H2 Bacteriophage vector Fig. 2. Cloning in a λ replacement vector. Molecular Biology

14 Plaques: 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. H2 Bacteriophage vector Molecular Biology

15 H2-2 M13 phage vector H2 Bacteriophage vector 1. Replication form (RF, dsDNA) of M13 phage can be purified and manipulated like a plamid. 2.Phage particles (ssDNA): DNA can be isolated in a single-stranded form. DNA sequencing.. Site-directed mutagenesis. Molecular Biology

16 M13 mp18 vector Molecular Biology

17 H3 COSMIDS, YACs AND BACs. Cloning large DNA fragments. Cosmid vectors. YAC vectors. Selection in S. cerevisiae. BAC vector Cloning vectors Molecular Biology

18 Analysis of eukaryotic genes and genome organization of eukaryotic requires vevtors with a larger capacity for cloned DNA than plasmids or phage λ. H3-1 Cloning large DNA fragments (Eukaryotic Genome project) H3 Cosmids and YACs Molecular Biology

19 H3-2 Cosmid vectors Cosmids 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. H3 Cosmids and YACs Molecular Biology

20 Formation of a cosmid clone Fig. 1. Formation of a cosmid clone. Molecular Biology

21 Yeast 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. H3-3 YAC vectors H3 Cosmids and YACs Molecular Biology

22 H3 Cosmids and YACs Molecular Biology

23 Selection 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. H3-4 Selection in S.cerevisiae H3 Cosmids and YACs Molecular Biology

24 H4 Eukaryotic Vectors 1. Shuttle vectors 2. Yeast episomal plasmids (Yeasts) 3. Agrobacterium tumefaciens Ti plasmid (Plants) 4. Baculovirus (Insects) 5. Mammalian viral vectors (Mammalian) Cloning vectors Molecular Biology

25 Shuttle vectors H4 Eukaryotic Vectors Molecular Biology

26 H4-1 Yeast episomal plasmids (YEps) H4 Eukaryotic Vectors Vectors for the cloning and expression of genes in Saccharomyces cerevisiae. Molecular Biology

27 Replicate as plasmid from 2m origin integrate by recombinantion YEp vector H4 Eukaryotic Vectors Molecular Biology

28 H4-2 Agrobacterium tumefaciens Ti plasmid H4 Eukaryotic Vectors Molecular Biology

29 crown gall or tumor H4 Eukaryotic Vectors Molecular Biology

30 Plant gene engineering using T-DNA vector H4 Eukaryotic Vectors Molecular Biology

31 H4-3 Baculovirus H4 Eukaryotic Vectors baculovirus is an insect virus which is used for the overexpression of animal proteins in insect cell culture. Molecular Biology

32 H4-4 Mammalian viral vectors H4 Eukaryotic Vectors Fig 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 273:113 Fig 2. retrovirus lifecycle Molecular Biology

33 Gene transfer H4 Eukaryotic Vectors Genes 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. Molecular Biology


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