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Section H – Cloning vectors
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 λ, λReplacement vectors, Packaging and
infection, Formation of plaques, λLysogens, 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
H1 Design of plasmid vectors —
Ligation products
• The most frequent unwanted product is
recreated vector plasmid formed by
circularization of the linear vector
fragment
Minipreparations from a
number of transformed
colonies, and screening by
digestion and agarose gel
electrophoresis.
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.
Screening by insertional inactivation of a resistance gene
Ampr
pBR322
ori
Tcr
B
B
B
Ampr
X
Recombinant
B
ori
X
B
Ampr
Tcr
Religated
ori
Ampicillin resistant?
yes
yes
Tetracycline resistant?
No
yes
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
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.
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.
lacZ encode enzyme b-galactosidase
lac promoter
(substrate of
X-gal the enzyme)
IPTG
Blue product
RegulatoryGene
Operon
i
p
o
z
y
a
DNA
m-RNA
Protein
β -Galactosidase
Transacetylase
Permease
Recreated vector: blue transformants
Recombinant plasmid: white transformants
(containing inserted DNA)
Recreated vector (no insert)
Recombinant plasmid (contain insert)
lacZ’: a shortened derivative of lacZ,
encoding N-terminal a-peptide of bgalactosidase.
Host strain carrying a mutant gene encoding
only the C-terminal portion of b -galactosidase
which can then complement the a-peptide to
produce the active enzyme
H1 Design of plasmid vectors —
Multiple cloning sites
Multiple restriction sites enable the convenient insertion of target DNA
into a vector
Lac promoter
MCS
Ampr
pUC18
(3 kb)
lacZ’
Insertion of
target DNA in
MCS
inactivates the
lacZ’
ori
SalI
SmaI
HincII
BamHI
EcoRI SacI KpnI XmaI
XbaI AccI PstI
SphI
…ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA…
. Thr Asn Ser Ser Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser…
Lac Z’
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 ⅡSK has promoters from
bacteriophages T7 and SP6 flanking an MCS.
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.
Promoter：
Strong Promoters
1.lacUV-5: a strong mutant lac promoter
independent of cAMP receptor protein (CRP or
CAP) .
2.lPL promoter
3.Phage T7 promoter
Fusion protein and fusion tag
1.Defined epitope : a small piece of peptide sequence
containing a defined epitopeor specific binding site
2.Green fluorescent protein : fusion with GFP.
3.His-tag: usually 6 consecutive histidines, which allows
purification of the fusion protein by binding to Ni 2+
column. Commonly used in E. coli.
Lac promoter
MCS
Ampr
pUC18
(3 kb)
lacZ’
ori
SalI
SmaI
HincII
BamHI
EcoRI SacI Kpn XmaI
XbaI AccI PstI SphI
…ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA…
I
. T h rA s n S er S e r Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser…
Lac Z’
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
How to make a fusion protein (in pUC18)?
SalI
SmaI
HincII
SacI
XmaI
BamHI
EcoRI
KpnI
XbaI AccI PstI SphI HindⅢ
ATGATTACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTT
M I T N S S S V P G D P L E S T C R H A S L
ATGATTACGAATTCGAGCTCGGTACCCGGGGATCCgatgcggagc..gtgaacggatagCTGCAG
M I T N S S S V P G D P M R S ..V N G *
From original ORF
Inserted ORF
Add one nucleotide (g) between BamHI site (GGATCC) and the
first codon (ATG) to fuse the two ORFs
BamHI: GGATCC
PstI: CTGCAG
CCTAGG
GACGTC
The following fusion is wrong:
ATGATTACGAATTCGAGCTCGGTACCCGGGGATCCatgcggagc..gtgaacggatagCTGCAG
M I T N S S S V P G D P C G G .. *
H2 Bacteriophage vectors —
Bacteriophage λ
1.Viruses that can
infect bacteria.
2.48.5 kb in length
3.Lytic phase：
Replicate and release
4.Lysogenic phase ：
integrate into host
genome
5.The phage  cos ends
（Linear or circular genome）
5‘-CGGGGCGGCGACCTCG-3’
3’-GCCCCGCCGCTGGAGC-5’
Circular form
Cleavage
(during packaging)
Ligation
(after infection)
GGGCGGGCGACCTCG-3’
5’-CG
+
GC-5’
3’-GCCCCGCCGCTGGA
Linear form
H2 Bacteriophage vectors —
λReplacement vectors
• e.g. EMBL3, DASH
•
Replace the nonessential region of
the phage genome with exogenous
DNA (~20kb)
H2 Bacteriophage vectors —
Packaging and infection
• Replication of phage λin vivo produces long linear molecules
with multiple copies of the λ genome. These concatemers are
then cleaved at the cos sites, to yield individual λ genomes,
which are then packaged into the phage particles.
• Ligated λ 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)
H2 Bacteriophage vectors —
Formation of plaques
1.
The clear areas within the lawn where lysis and reinfection have prevented the cells from growing.
2. Recombinant l DNA may be purified from phage
particles from plaques or from liquid culture.
H2 Bacteriophage vectors —
λLysogens
• 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.
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.
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.
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.
Cloning

RF
like plasmid
Transfection
recombinant
DNA

Growth
 Plaques formation
plating on
slow
a cell lawn
growth
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.
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.
• 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.
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
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.
Cloning in a cosmid
vector C2XB
H3 Cosmids, YACs and BACs —
Essential components of
YAC vectors :
• Centromere
• Telomere
• Autonomous replicating
sequence
• Ampr for selective
amplification and
markers
YAC vectors
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.
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
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
Cosmid：35-45 kb
YAC: > 1Mb (1987)
H4 Eukaryotic vectors —
Cloning in eukaryotes
• Many applications of genetic engineering
require vectors for the expression of genes
in diverse eukaryotic species.
H4 Eukaryotic vectors —
Transfection of eukaryotic cells
The take-up of DNA into eukaryotic cells
1. 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.
2. Much lower efficiency
Transfection methods:
•Calcium phosphate
•Electroporation
•Gene gun
•Microinjection
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.
A Shuttle vector
MCS
H4 Eukaryotic vectors —
Yeast episomal plasmids
Vectors for the cloning and expression of
genes in Saccharomyces cerevisiae.
1.
•
•
•
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.
YEp vector
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.
Agrobacterium mediated gene transfer
crown gall or tumor
Agrobacterium mediated gene transfer
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.
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
H4 Eukaryotic vectors —
Direct gene transfer
• Genes may be transiently or
permanently introduced into cultured
eukaryotic cells without the use of
vector.
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.
3. Infection of E. coli by bacteriophage λis 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 λvector.
C a BAC.
D a YAC.
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.
THANK YOU !