Section H

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Molecular Biology Course

• Section H
• Cloning Vectors

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Introduction
vectors
Cloning vectors to clone a gene in a
vector Expression vectors allowing the
exogenous DNA to be inserted, stored, and
manipulated mainly at DNA level Integration
vectors allowing the exogenous DNA to be
inserted, stored, and expressed.
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Cloning vectors
H1 Plasmid vecters H2 Bacteriophage vectors H3
Cosmids and YACs H4 Eukaryotic vectors
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Cloning vectors
H1 Design of Plasmid Vectors
H1-1 A plasmid vector for cloning H1-2 A
plasmid vector for gene expression
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H1 Design of Plasmid Vectors
H1-1 A plasmid vector for cloning

• autonomously replicating independent of hosts
  genome.
• Easily to be isolated from the host cell.
  (Plasmid preparation)
• Selective markers Selection of cells
• (1) Containing vector one marker is enough
  (2) Containing ligation products
• twin antibiotic resistance
• blue-white screening
• 4. Contains a multiple cloning site (MCS)

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Screening by insertional inactivation of a
resistance gene
B X B
B
Tcr
Ampr
Ampr
X
B
ori
ori
Ampicillin resistant? yes
yes Tetracycline resistant? No
yes
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H1 Design of Plasmid Vectors
Replica plating transfer of the colonies from
one plate to another using absorbent pad or
Velvet (??).
transfer of colonies
ampicillin tetracycline
ampicillin
these colonies have bacteria with recombinant
plasmid
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H1 Design of Plasmid Vectors
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H1 Design of Plasmid Vectors
Blue white screening
Screening by insertional inactivation of the lacZ
gene
Lac promoter
MCS (Multiple cloning sites, ?????)
Ampr
pUC18 (3 kb)
lacZ
ori
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.
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H1 Design of Plasmid Vectors
lacZ encode enzyme b-galactosidase
(substrate of the enzyme)
lac promoter?
X-gal
IPTG
Blue product
The expression of active b-galactosidase has to
be vector dependent for the selection purpose
lacZ a shortened derivative of lacZ, encoding
N-terminal a-peptide of b-galactosidase. Host
strain for vectors containing lacZ contains a
mutant gene encoding only the C-terminal portion
of b-galactosidase which can then complement the
a-peptide to produce the active enzyme
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H1 Design of Plasmid Vectors
Recreated vector blue transformants Recombinant
plasmid containing inserted DNA white
transformants
Recreated vector (no insert)
Recombinant plasmid (contain insert)
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H1 Design of Plasmid Vectors
Multiple cloning sites
Multiple restriction sites enable the convenient
insertion of target DNA into a vector
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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.

1. Promoter and terminator for RNA transcription are
   required.
2. Intact ORF and ribosomal binding sites (RBS) are
   required for translation.

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H1 Design of Plasmid Vectors

• Some cloning vector can be used to transcribe a
  gene. (pUC vectors)
• Special transcriptional vectors pGEM series
  containing promoters from bacteriophages T7 and
  SP6 for in vitro transcription with the
  corresponding polymerases.
• Expression vector (transcription
  translation).

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H1 Design of Plasmid Vectors

1. The ORF of the inserted gene has to be in the
   same direction as that of the lacZ
2. A fusion protein contains the N-terminal sequence
   of lacZ and the inserted ORF will be produced

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H1 Design of Plasmid Vectors
Expression vector (transcription translation).

• Promoters
• lacUV-5 a mutant lac promoter which is
  independent of cAMP receptor protein.
  (constitutive expression)
• lPL promoter
• Phage T7 promoter

Fused proteins
Individual proteins
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H1 Design of Plasmid Vectors
T7 promoter
RBS
Start codon
MCS
Ampr
Transcription terminator
T7 expression vector
ori
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H1 Design of Plasmid Vectors
Fused proteins
Lac fusions (discussed) His-tag fusions A
sequence encodes His-tag was inserted at the N-
terminus of the target ORF, which allows
purification of the fusion protein to be purified
by binding to Ni2 column.
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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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H2 Bacteriophage vector
? 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)
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H2 Bacteriophage vector
H2-1? phage
Protein coat
DNA
Long (left) arm
short (right) arm
cos
cos
Nonessential region
Exogenous DNA (20-23 kb)
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H2 Bacteriophage vector
The phage ? cos ends
5-CGGGGCGGCGACCTCG-3 3-GCCCCGCCGCTGGAGC-5
Cleavage
Ligation (during packaging)
(after infection)
GGGCGGGCGACCTCG-3 5-CG
GC-5 3-GCCCCGCCGCTGGA
Circular form
Linear form
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H2 Bacteriophage vector
? phage
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H2 Bacteriophage vector
? replacement vector

• Replace the nonessential region of the phage
  genome with exogenous DNA ( 20 kb)
• high transformation efficiency (1000-time higher
  than plasmid)

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? replacement vector
H2 Bacteriophage vector
2. Packing with a mixture of the phage coat
proteins and phage DNA-processing enzymes

1. Ligation

3. Infection and formation of plaques
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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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H2 Bacteriophage vector

• lysogens
• in cloning techniques

Genes or foreign sequences may be incorporated
essentially permanently into the genome of E.coli
by integration of a ? vector containing the
sequence of interest.
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H2 Bacteriophage vector
H2-2 M13 phage

• A filamentous phage
• Phage particles contain a 6.7kb circular single
  strand of DNA.
• After infection of a sensitive E.coli host,the
  complementary strand is synthesized,and the DNA
  replicated as a double-stranded circle,the
  replicative form(RF) with about 100 copies per
  cell.
• Contrasting to phage ?,the cell are not lysed by
  M13,but continue to grow slowly,and
  single-stranded forms are continuously packaged
  and released from the cells as new phage
  particles.

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H2 Bacteriophage vector
M13 phage vectors

• 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 (Topic J2)
• Site-directed mutagenesis (Topic J5)

Cloning (RF, like plasmid) ? transfection
(recombinant DNA) ? growth (plating on a cell
lawn) ? plaques formation (slow growth)
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Blue-white selection
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H2 Bacteriophage vector
Hybrid plasmid-M13 vectors

• Small plasmid vectors (pBluescript) being
  developed to incorporate M13 functionality
• Contain both the plasmid and M13 origin of
  replication
• Normally propagate as true plasmids
• Can be induced to form single-stranded phage
  particles by infection of the host cell with a
  helper phage.

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Cloning vectors
H3 Cosmids and YACs
H3-1 Cloning large DNA fragments (gt 20 kb) H3-2
Cosmid vectors H3-3 YAC vectors H3-4 Selection in
S. cerevisiae (????)
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H3 Cosmids and YACs
H3-1 Cloning large DNA fragments
(Eukaryotic Genome project)
Analysis of eukaryotic genes and the genome
organization of eukaryotes requires vectors with
a larger capacity for cloned DNA than plasmids or
phage ?. Human genome (3 x 109 bp) large genome
and large gene demand vectors with a large size
capacity.
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H3 Cosmids and YACs
H3-2 Cosmid vectors

1. Utilizing the properties of the phage l cos sites
   in a plasmid vector.
2. A combination of the plasmid vector and the COS
   site which allows the target DNA to be inserted
   into the l head.
3. The insert can be 37-52 kb.

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Formation of a cosmid clone
C) Packaging and infect
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B
H3 Cosmids and YACs
cos
SmaI
Cloning in a cosmid vector
B
S
B
S
cos
cos
B
cos
B
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H3 Cosmids and YACs
H3-3YAC vectors
Can accommodate genomic DNA fragments of more
than 1 Mb, and can be used to clone the entire
human genome, but not good in mapping and analysis

• Essential components of YAC vectors
• Centromers (CEN), telomeres (TEL) and
  autonomous replicating sequence (ARS) for
  proliferation in the host cell.
• ampr for selective amplification and markers
  such as TRP1 and URA3 for identifying cells
  containing the YAC vector in yeast cells.
• Recognition sites of restriction enzymes (e.g.,
  EcoRI and BamHI)

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H3 Cosmids and YACs
Yeast selection
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H3 Cosmids and YACs
Cloning in YAC vector
Digest with BamHI/SnaI
Ligate with blunt ends
Transfect into yeast
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H3 Cosmids and YACs
H3-4 Selection in S.cerevisiae

• Saccharomyces cerevisiae selectable markers do
  not confer resistance to toxic substances
• Growth of yeast on selective media lacking
  specific nutrients can serve for selection.
  Auxotrophic yeast mutants (?????) are made as
  host strains for plasmids containing the genes
  complementary to the growth defect .
• For example TRP1 mutants cant make
  tryptophan, and can only grow on media
  supplemented with tryptophan. The presence of a
  plasmid containing gene encoding tryptophane
  enables the cell to grow on media without
  tryptophan.

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Cloning vectors
H4 Eukaryotic Vectors

• Transfection of eukarotic cells
• Shuttle vectors
• H4-1 Yeast episomal plasmids (Yeasts)
• H4-2 Agrobacterium tumefaciens (???) Ti plasmid
  (Plants)
• H4-3 Baculovirus (Insects)
• H4-4 Mammalian viral vectors (Mammalian)

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H4 Eukaryotic Vectors
Transfection
The take-up of DNA into eukaryotic cells

• more problematic than bacterial transformation
• Much lower efficiency in the progress
• Transfection methods
• Electroporation
• Microinjection
• liposome

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H4 Eukaryotic Vectors
Shuttle vectors
Vectors contain sequences required for
replication and selection in both E. coli and the
desired host cells, so that the construction and
many other manipulation of the recombinant
plasmids can be completed in E. coli. Most of
the eukaryotic vectors are constructed as shuttle
vectors
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H4 Eukaryotic Vectors
A Shuttle vector
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H4 Eukaryotic Vectors
H4-1 Yeast episomal plasmids (YEps)
Vectors for the cloning and expression of genes
in Saccharomyces cerevisiae.

• Based on 2 micron (2m) plasmid which is 6 kb in
  length.
• One origin
• Two genes involved in replication
• A site-specific recombination protein FLP,
  homologous to l Int.
• 2. Normally replicate as plasmids, and may
  integrate into the yeast genome.

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A YEp vector
H4 Eukaryotic Vectors
MCS
Insert Figure 1
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H4 Eukaryotic Vectors
Replicate as plasmid from 2m origin
YEp vector
integrate by recombinantion
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H4 Eukaryotic Vectors
H4-2 Agrobacterium tumefaciens Ti plasmid
T-DNA
Ti plasmid 200kb
plant chromosome
Integrated T-DNA
Gene induce crown gall
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H4 Eukaryotic Vectors
crown gall or tumor
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H4 Eukaryotic Vectors
Recombinant Ti plasmid

1. Place the target gene in the T-DNA region of a Ti
   plasmid, then transform the recombinant Ti
   plasmid. (WT is not good because of the crown
   gall formation)
2. Recombinant T-DNA transformed into the A.
   tumefaciens cell carrying a modified Ti plasmid
   without T-DNA (T-DNA that are responsible for
   crown gall formation. The deleted T-DNA is called
   disarmed T-DNA shuttle vector).

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H4 Eukaryotic Vectors
Plant gene engineering using T-DNA vector
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H4 Eukaryotic Vectors
H4-3 Baculovirus

• Infects insect cells
• The strong promoter expressing polyhedrin protein
  can be used to over-express foreign genes
  engineered. Thus, large quantities of proteins
  can be produced in infected insect cells.
• Insect expression system is an important
  eukaryotic expression system.

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H4 Eukaryotic Vectors
H4-4 Mammalian viral vectors

• SV40 5.2 kb, can pack DNA fragment similar to
  phage l.
• Retroviruss
• single-stranded RNA genome, which copy to dsDNA
  after infection.
• Have some strong promoters for gene expression
• Gene therapy

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H4 Eukaryotic Vectors
Gene transfer

• Genes may be transiently or permanently
  introduced into cultured eukaryotic cells without
  the use of vector in strict sense.
• Transient expression
• Integration