Section H

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Molecular Biology
Molecular Biology

• Section H
• Cloning Vectors

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Molecular Biology
Cloning vectors
Content
. DESIGN OF PLASMID VECTORS . BACTERIOPHAGE
VECTORS . COSMIDS, YACs AND BACs . EUKARYOTIC
VECTORS
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Cloning vectors
H1 Design of Plasmid Vectors
Fig. 1. (a) Screening by insertional inactivation
of a resistance gene (b) replica plating.
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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
bluewhite screening (b) the colonies produced
by bluewhite screening.
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Molecular Biology
Cloning vectors
H1 Design of Plasmid Vectors
Fig. 3. A multiple cloning site at the 5'-end of
lacZ'
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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.

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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
Individual proteins
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Molecular Biology
H1 Design of Plasmid Vectors
Fig. 4. A plasmid designed for expression of a
gene using the T7 system
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Cloning vectors
H2 Bacteriophage vector

Tow examples H2-1 ? phage
bacteriophage? ? replacement vector
H2-2 M13 phage M13 phage vector
Cloning in M13 Hybrid plasmid-M13 vectors
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Molecular Biology
? 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 ? and its genome (b) the phage
? cos ends.
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Molecular Biology
H2 Bacteriophage vector
? replacement vector

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

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H2 Bacteriophage vector
Protein coat
Fig. 2. Cloning in a ? replacement vector.
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H2 Bacteriophage vector
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.
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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.

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Molecular Biology
M13 mp18 vector
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Cloning vectors
H3 COSMIDS, YACs AND BACs
. Cloning large DNA fragments . Cosmid
vectors . YAC vectors . Selection in S.
cerevisiae . BAC vector
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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 ?.
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Molecular Biology
H3 Cosmids and YACs
H3-2 Cosmid vectors
Cosmids use the ? packaging system to
package large DNA fragments bounded by ? 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 ? particles. Cosmids have a
capacity for cloned DNA of 30-45 kb.
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Formation of a cosmid clone
Fig. 1. Formation of a cosmid clone.
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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.
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Molecular Biology
H3 Cosmids and YACs
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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.
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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)
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H4 Eukaryotic Vectors
Shuttle vectors
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H4 Eukaryotic Vectors
H4-1 Yeast episomal plasmids (YEps)
Vectors for the cloning and expression of genes
in Saccharomyces cerevisiae.
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Replicate as plasmid from 2m origin
H4 Eukaryotic Vectors
YEp vector
integrate by recombinantion
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H4 Eukaryotic Vectors
H4-2 Agrobacterium tumefaciens Ti plasmid
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H4 Eukaryotic Vectors
crown gall or tumor
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H4 Eukaryotic Vectors
Plant gene engineering using T-DNA vector
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H4 Eukaryotic Vectors
H4-3 Baculovirus
baculovirus is an insect virus which is used for
the overexpression of animal proteins in insect
cell culture.
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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 273113
Fig 2. retrovirus lifecycle
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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.