Section H

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Section H Cloning vectors
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Contents

• H1 Design of plasmid vectors
• Ligation products, Twin antibiotic
  resistance, Blue-white screening, Multiple
  cloning sites, Transcription of cloned inserts,
  Expression vectors
• H2 Bacteriophage vectors
• Bacteriophage ?, ?Replacement vectors,
  Packaging and infection, Formation of plaques,
  ?Lysogens, M13 phage vectors, Cloning in M13,
  Hybrid plasmid-M13 vectors
• H3 Cosmids, YACs and BACs
• Cloning large DNA fragments, Cosmid vectors,
  YAC vectors, Selection in S. cerevisiae, BAC
  vectors
• H4 Eukaryotic vectors
• Cloning in eukaryotes, Transfection of
  eukaryotic cells, Shuttle vectors, Yeast episomal
  plasmids, Agrobacterium tumefaciens TI plasmid,
  Baculovirus, Mammalian viral vectors, Direst gene
  transfer

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H1 Design of plasmid vectors
Ligation products

• The most frequent unwanted product is recreated
  vector plasmid formed by circularization of the
  linear vector fragment

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Minipreparations from a number of
transformed colonies, and screening by digestion
and agarose gel electrophoresis.
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H1 Design of plasmid vectors
Twin antibiotic resistance

• Contain two antibiotic resistance genes
• If a target DNA fragment is ligated into
  the coding region of one of the resistance genes
  the gene will become insertionally inactivated,
  and can be determined by the antibiotic
  resistance exhibited by the transformants.

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Screening by insertional inactivation of a
resistance gene
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Replica plating transfer of the colonies from
one plate to another using absorbent pad or velvet
transfer of colonies
ampicillin
ampicillin tetracycline
These colonies have bacteria with recombinant
plasmid
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H1 Design of plasmid vectors
Blue-white screening

• A more sophisticated procedure can be carried out
  on a single transformation plate
• Blue white screening
• Involves the insertional inavtivation of the gene
  lacZ.

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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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Recreated vector blue transformants Recombinant
plasmid white transformants (containing inserted
DNA)
Recreated vector (no insert)
Recombinant plasmid (contain insert)
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lacZ a shortened derivative of lacZ,
encoding N-terminal a-peptide of b-galactosidase.
Host strain carrying a mutant gene encoding
only the C-terminal portion of ? -galactosidase
which can then complement the a-peptide to
produce the active enzyme
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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
Transcription of cloned inserts

• Some cloning vector The pUC vectors have a
  promoter (lac) adjacent to the site of insertion
  of a cloned fragment, such a promoter could be
  used to transcribe the inserted DNA, either to
  produce an RNA transcript in vitro (used as a
  hybridization probe), or to express the protein
  product of a gene.
• Special transcriptional vectors
• The pBluescript ?SK has promoters from
  bacteriophages T7 and SP6 flanking an MCS.

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H1 Design of plasmid vectors
Expression vectors

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

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• Strong Promoters
• Promoter
• lacUV-5 a strong mutant lac promoter
  independent of cAMP receptor protein (CRP or CAP)
  .
• lPL promoter
• Phage T7 promoter

• Fusion protein and fusion tag
• Defined epitope a small piece of peptide
  sequence containing a defined epitopeor specific
  binding site
• Green fluorescent protein fusion with GFP.
• His-tag usually 6 consecutive histidines, which
  allows purification of the fusion protein by
  binding to Ni 2 column. Commonly used in E.
  coli.

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

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How to make a fusion protein (in pUC18)?
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H2 Bacteriophage vectors
Bacteriophage ?
1.Viruses that can infect bacteria. 2.48.5 kb in
length 3.Lytic phaseReplicate and
release 4.Lysogenic phase integrate into host
genome
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5.The phage ? cos ends (Linear or circular genome)
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 vectors
?Replacement vectors

• e.g. EMBL3, ?DASH
• Replace the nonessential region of the phage
  genome with exogenous DNA (20kb)

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H2 Bacteriophage vectors
Packaging and infection

• Replication of phage ?in vivo produces long
  linear molecules with multiple copies of the ?
  genome. These concatemers are then cleaved at the
  cos sites, to yield individual ? genomes, which
  are then packaged into the phage particles.
• Ligated ? ends which do not contain an insert, or
  have one which is smaller or larger than the 20kb
  optimum, are too small or to large to be
  packaged, and recombinants with two left or right
  arms are likewise not viable.
• High infection efficiency (109 recombinants/ug
  vector DNA, 100-time higher than plasmid)

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H2 Bacteriophage vectors
Formation of plaques

1. The clear areas within the lawn where lysis and
   re-infection have prevented the cells from
   growing.
2. Recombinant l DNA may be purified from phage
   particles from plaques or from liquid culture.

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H2 Bacteriophage vectors ?Lysogens

• 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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The E. coli strain BL21(DE3) include the gene
for T7 RNA polymerase under control of the lac
promoter as a ? lysogen. The gene can be induced
by IPTG, and the polymerase will then transcribe
the gene in the expression vector.
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H2 Bacteriophage vectors
M13 phage vectors

• E. coli vector
• 6.7 kb circular single strand of DNA
• 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 vectors Cloning in M13

• When the single-stranded form of a fragment is
  required fragments are subcloned into M13 RF
  using standard plasmid methods.

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H2 Bacteriophage vectors Hybrid
plasmid-M13 vectors

• Small plasmid vectors being developed to
  incorporate M13 functionality.
• Contain both the plasmid and M13 origins 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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H3 Cosmids, YACs and BACs Cloning
large DNA fragments

• 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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• The limitation of conventional gel
  electrophoresis large DNA fragments do not
  separate, but instead, comigrate, because nucleic
  acids alternate between globular and linear
  forms.
• If the field is applied discontinuously and
  the direction is also made to vary,the DNA
  molecules reorient their long axes and takes
  longer for larger molecules.

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H3 Cosmids, YACs and BACs
Cosmid vectors

• Utilizing the properties of the phage l cos sites
  in a plasmid vector.
• The insert can be 30-45 kb

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The simplest cosmid vector A normal small
plasmid, containing a plasmid origin of
replication, a selectable marker, a cos site, a
suitable restriction site for cloning.
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Cloning in a cosmid vector C2XB
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H3 Cosmids, YACs and BACs YAC vectors

• Essential components of YAC vectors
• Centromere
• Telomere
• Autonomous replicating sequence
• Ampr for selective amplification and markers

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YACs can accommodate genomic DNA fragments of
more than 1 Mb, and can be used to clone the
entire human genes, but not good in mapping and
analysis. YACs have been invaluable in
mapping the large-scale structure of large
genomes, for example in the Human Genome Project.
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H3 Cosmids, YACs and BACs
Selection in S. cerevisiae

• (1)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.
• (2)Saccharomyces cerevisiae selectable markers do
  not confer resistance to toxic substances

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H3 Cosmids, YACs and BACs BAC vectors

• BAC Bacterial Artificial Chromosome, 300kb

Vectors for large DNA fragments BAC 300kb PAC
Bacteriophage P1 cloning system,100kb Cosmid35-45
kb YAC gt 1Mb (1987)
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H4 Eukaryotic vectors
Cloning in eukaryotes

• Many applications of genetic engineering require
  vectors for the expression of genes in diverse
  eukaryotic species.

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H4 Eukaryotic vectors Transfection of
eukaryotic cells

• The take-up of DNA into eukaryotic cells
• More problematic than bacterial transformation.
  The plant cell wall must normally be digested to
  yield fragile protoplasts, which may then take up
  DNA fairly readily.
• Much lower efficiency

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• Transfection methods
• Calcium phosphate
• Electroporation
• Gene gun
• Microinjection

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H4 Eukaryotic vectors Shuttle vectors

• Most of the eukaryotic vectors are constructed as
  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 vectors can be
  completed in E. coli., before transfer to the
  appropriate eukaryotic cells.

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A Shuttle vector
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H4 Eukaryotic vectors Yeast
episomal plasmids

• 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 ? Int.
• 2. Normally replicate as plasmids, and may
  integrate into the yeast genome.

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YEp vector
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H4 Eukaryotic vectors Agrobacterium
tumefaciens Ti plasmid

• 1. Place the target gene in the T-DNA region of
  a Ti plasmid, then transform the recombinant Ti
  plasmid. (Not good because of the crown gall
  formation)
• Deletion of the genes in T-DNA that are
  responsible for crown gall formation. The deleted
  T-DNA is called disarmed T-DNA shuttle vector.
• 2. The T-DNA and the remainder of the Ti plasmid
  are on separate molecules within the same
  bacterial cell, integration will still take
  place. Plasmid with recombinant T-DNA can be
  transformed into the A. tumefaciens cell carrying
  a modified Ti plasmid without T-DNA.

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Agrobacterium mediated gene transfer
crown gall or tumor
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Agrobacterium mediated gene transfer
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H4 Eukaryotic vectors Baculovirus

1. Infects insect cells
2. 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.
3. Insect expression system is an important
   eukaryotic expression system.

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H4 Eukaryotic vectors
Mammalian viral vectors

• 1. SV40
• 5.2 kb, suffers from packaging constraints
  similar to phage l.
• 2. Retrovirus
• Single-stranded RNA genome, which copy to
  dsDNA after infection. Integrated into the host
  genome by a transposition-like mechanism.
• Have some strong promoters for gene
  expression. Considered as vectors for gene
  therapy

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H4 Eukaryotic vectors
Direct gene transfer

• Genes may be transiently or permanently
  introduced into cultured eukaryotic cells without
  the use of vector.

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Multiple choice questions

• 1. Blue-white selection is used .
• A to test for the presence of a plasmid in
  bacteria.
• B to reveal the identity of a cloned DNA
  fragment.
• C to express the product of a cloned gene.
• D to test for the presence of a cloned insert
  in a plasmid.
• 2. A multiple cloning site .
• A contains many copies of a cloned gene.
• B allows flexibility in the choice of
  restriction enzymes for cloning.
• C allows flexibility in the choice of organism
  for cloning.
• D contains many copies of the same restriction
  enzyme site.

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• 3. Infection of E. coli by bacteriophage ?is
  normally detected by .
• A resistance of the bacteria to an antibiotic.
• B the growth of single bacterial colonies on an
  agar plate.
• C the appearance of areas of lysed bacteria on
  an agar plate.
• D restriction digest of the bacterial DNA.
• 4. Which vector would be most appropriate for
  cloning a 150 kb fragment of DNA?
• A a plasmid.
• B a ?vector.
• C a BAC.
• D a YAC.

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• 5. Which vector would you chose to express a
  foreign gene in a plant?
• A a baculovirus vector.
• B a retroviral vector.
• C a Yep vector.
• D a T-DNA vector.

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