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Molecular Biology. Molecular Biology. Section H Cloning Vectors. Molecular Biology. Cloning vectors. Content. . DESIGN OF PLASMID VECTORS . BACTERIOPHAGE VECTORS . COSMIDS, YACs AND BACs . EUKARYOTIC VECTORS . Molecular Biology. Cloning vectors. H1 Design of Plasmid Vectors.

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Presentation Transcript
slide1

Molecular Biology

Molecular Biology

Section H

Cloning Vectors

slide2

Molecular Biology

Cloning vectors

Content

. DESIGN OF PLASMID VECTORS

. BACTERIOPHAGE VECTORS

. COSMIDS, YACs AND BACs

. EUKARYOTIC VECTORS

slide3

Molecular Biology

Cloning vectors

H1 Design of Plasmid Vectors

Fig. 1. (a) Screening by insertional inactivation of a resistance gene; (b) replica plating.

slide4

Molecular Biology

Cloning vectors

H1 Design of Plasmid Vectors

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.

Fig. 2. (a) A plasmid vector designed for blue–white screening; (b) the colonies produced by blue–white screening.

slide5

Lac promoter

MCS (Multiple cloning sites,

多克隆位点)

Ampr

pUC18

(3 kb)

lacZ’

ori

Molecular Biology

Cloning vectors

H1 Design of Plasmid Vectors

Fig. 3. A multiple cloning site at the 5′-end of lacZ′

slide6

Molecular Biology

H1 Design of Plasmid Vectors

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.
slide7

Molecular Biology

H1 Design of Plasmid Vectors

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

Individualproteins

slide8

Molecular Biology

H1 Design of Plasmid Vectors

Fig. 4. A plasmid designed for expression of a gene using the T7 system

slide9

Molecular Biology

Cloning vectors

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

slide10

Molecular Biology

λ phage

H2 Bacteriophage vector

.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.

slide11

Molecular Biology

H2 Bacteriophage vector

λ replacement vector

  • . Replace the nonessential region of the phage genome with exogenous DNA
  • . high transformation efficiency (1000-time higher than plasmid)
slide12

Molecular Biology

H2 Bacteriophage vector

Protein coat

Fig. 2. Cloning in a λ replacement vector.

slide13

Molecular Biology

H2 Bacteriophage vector

Plaques:the clear areas within the lawn where lysis and re-infection have prevented the cells from growing.

Recombinant l DNAmay be purified from phage particles from plaques or from liquid culture.

slide14

Molecular Biology

H2 Bacteriophage vector

H2-2 M13 phage vector

  • Replication 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.
slide15

Molecular Biology

M13 mp18 vector

slide16

Molecular Biology

Cloning vectors

H3 COSMIDS, YACs AND BACs

. Cloning large DNA fragments

. Cosmid vectors

. YAC vectors

. Selection in S. cerevisiae

. BAC vector

slide17

Molecular Biology

H3 Cosmids and YACs

H3-1 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.

slide18

Molecular Biology

H3 Cosmids and YACs

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 30-45 kb.

slide19

Molecular Biology

Formation of a cosmid clone

Fig. 1. Formation of a cosmid clone.

slide20

Molecular Biology

H3 Cosmids and YACs

H3-3 YAC vectors

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.

slide21

Molecular Biology

H3 Cosmids and YACs

slide22

Molecular Biology

H3 Cosmids and YACs

H3-4 Selection in S.cerevisiae

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.

slide23

Molecular Biology

Cloning vectors

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)

slide24

Molecular Biology

H4 Eukaryotic Vectors

Shuttle vectors

slide25

Molecular Biology

H4 Eukaryotic Vectors

H4-1 Yeast episomal plasmids (YEps)

Vectors for the cloning and expression of genes in Saccharomyces cerevisiae.

slide26

Molecular Biology

Replicate as plasmid from 2m origin

H4 Eukaryotic Vectors

YEp vector

integrate by recombinantion

slide27

Molecular Biology

H4 Eukaryotic Vectors

H4-2 Agrobacterium tumefaciens Ti plasmid

slide28

Molecular Biology

H4 Eukaryotic Vectors

crown gall or tumor

slide29

Molecular Biology

H4 Eukaryotic Vectors

Plant gene engineering using T-DNA vector

slide30

Molecular Biology

H4 Eukaryotic Vectors

H4-3 Baculovirus

baculovirus is an insect virus which is used for the overexpression of animal proteins in insect cell culture.

slide31

Molecular Biology

H4 Eukaryotic Vectors

H4-4 Mammalian viral 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

slide32

Molecular Biology

H4 Eukaryotic Vectors

Gene transfer

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.