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Section H
Host and Vector
H1
H2
H3
H4
Section H: Cloning Vectors
E.coli/Plasmid Vectors
E.coli/Bacterophage Vectors
Yeast/YAC and E.coli/BAC
Eukaryotic Host/Vectors
Yang Xu, College of Life Sciences
H1 E.coli/Plasmid Vectors
•
•
•
•
•
E.coli/pBR322 plasmid
E.coli/pUC plasmid vectors
Multiple cloning sites
E.coli/pGEM
E.coli/T7 Expression vectors
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Ligation products
E
A
B
Ligation products:
• Recombinant plasmid: With a target fragment.
• Recreated vectors: When ligating a target fragment into a
plasmid vector, the most frequent unwanted product is the
recreated vector plasmid
Screening of ligation products:
• Agarose gel electrophoresis: For mini-preparations from a
number of transformed colonies. Screening by digestion
and agarose gel electrophoresis;
• Specially developed vectors: For large scale preparations.
Now more efficient methods based on specially
developed vectors have been devised (see below).
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
E.coli/pBR322 plasmid
Mechanisms--Insertional inactivation of the resistance genes: If
a target DNA fragment is ligated into the coding region of tet
A, the gene will become insertionally inactivated.
B
ampr
pBR322
B
X
Ori
X
ampr
+
+
B
tetA
B
B
Section H: Cloning Vectors
B
tetA
Ori
Yang Xu, College of Life Sciences
Twin antibiotic resistance screening
1.
•
•
2.
Transformant plating:
Recombinant: can only grow in ampicillin plates;
Recreated vectors: can grow in ampicillin and tetracycline plates
Replica plating: The colonies grown on a normal ampicillin
plate are transferred, using an absorbent pad, to a second plate
containing tetracycline.
Recombinant
transfer
Comparison
Ampicillin only
Section H: Cloning Vectors
Ampicillin and tetracycline
Yang Xu, College of Life Sciences
Blue-white screening
• Example--pUC18 plasmid: This one contains an ampr and a lac
Z gene, which encodes the -galactosidase, and is under the
control of the lac promoter.
lac promoter
ampr
pUC18
Ori
MCS
lacZ’
Blue: no insert
White: insert
ampr/X-gal plate
• Mechanisms--Insertional inactivation of the lac Z gene:
Under the effect of -galactosidase, the substrate X-gal will
produce a blue product.
1. The blue colonies: probably contain recreated vector.
2. The white colonies: have no expressed -galactosidase and
are hence likely to contain the inserted target fragment.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Multiple cloning sites
• The first vectors which used blue-white selection also pioneered
the application of multiple cloning site (MCS).
Definition: The pUC series contain an engineered lacZ‘ gene,
which has multiple restriction enzyme sites within the first part
of the coding region of the gene, which is known as “MCS”.
Function: The insertion of target DNA in any of these sites, will
inactivates the lac Z’ gene, to give a white colony.
SmaI
EcoRI
SacI
KpnI XmaI BamHI
AccI HincII
XbaI
SalI
PstI
SphI
HindIII
GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCT
LacZ’
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
E.coli/T7 expression vectors
• Definition of expression vectors:
Cloned geneexpression vector
T7
hostfusion protein.
• Structure
– T7 promoter: a strong promoter;
– RBS: ribosome binding site;
– ATG: translation initiation condon
– MCS: Multiple cloning sites
– TT: transcription terminator.
– ampr,. ori,
• His-tag: Some expression vectors are
designed to have six histidine codons that
encode a hexahistidine tag at the N
terminus of the expressed protein, which
allows one-step purification on an affinity
column containing Ni2+.
Section H: Cloning Vectors
RBS
MCS
TT
ATG
T7
expressional
vector
Yang Xu, College of Life Sciences
H2 Bacterophage Vectors
•
•
•
•
•
•
Bacteriophage 
E.coli/ Replacement vectors
E.coli/Cosmid vectors
E.coli/M13 phage vectors
E.coli/pBluescript vectors
Hybrid plasmid-M13 vectors
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Bacteriophage  (life cycle)
Process of phage  infecting E. coli: In brief,
1. Phage injects its linear DNA into E.coli, then ligates into a circle.
2. The circle DNA may replicate to form many “phage particles”,
3. which  are released from the cell by lysis and cell death (lytic
phase),  or integrate into the host genome (lysogenic phase).
Lytic life
Section H: Cloning Vectors
Lysogenic
life
Yang Xu, College of Life Sciences
Bacteriophage 
Coat protein
Liner DNA
Phage 
5’-CGGGGCGGCGACCTCG-3’
3’-GCCCCGCCGCTGGAGC-5’
5’-CG
GGGCGGCGACCTCG-3’
3’-GCCCCGCCGCTGGA
GC-5’
Cos end
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
E.coli/ Replacement vectors
Examples: EMBL3 and  DASH.
A representative scheme for cloning:
Long
1. The  vector DNA is cleaved with
BamH1 and the long (19 kb) and
short (9 kb) arms (p116 Fig. 1) are
purified;
2. The target fragments are prepared
by digestion, also with BamH1 or a
compatible enzyme (Sau3A);
3. The target fragments are treated
with alkaline phosphatase to
B
prevent them ligating to each other;
4. The  arms and the target
fragments are ligated together at
relatively high concentration to
Can not
form long linear products.
Parking
Section H: Cloning Vectors
arm
Short
arm
Replace.
48.5 kb
B
B
 20kb
infect
E.coli
Yang Xu, College of Life Sciences
Packaging and infection
The Recombinants that can not be packaged:
1. Ligated  ends which do not contain an insert;
2. The insert is much smaller or larger than the 20 kb;
3. The recombinants with two left or right arms.
Packaging:
in vivo
B
Replication concata-mers
cleave
Section H: Cloning Vectors
individual  genomes
in vitro
A mixture of phage coat proteins and
the phage DNA-processing enzymes
Packaging
Infection of E. coli
phage
109 recombinants per
particles
mg of vector DNA.
Yang Xu, College of Life Sciences
Formation of plaques
Plaques are the analogs of single bacterial colonies.
Formation:
The infected E.coli cells from a packaging reaction are spread on an
agar plate,
The plate has been pre-spread with uninfected cells, which will
grow to form a continuous lawn.
After incubation, phage-infected cells result in clear areas, that are
plaques, where cycles of lysis and re-infection have prevented the
cells from growing.
E.coli lawn
Plaques
Recombinant  DNA may be purified:
• from phage particles isolated from plaques or
• from the supernatant of a culture infected
with a specific recombinant plaque.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
E.coli/Cosmid vectors
• Structure:
1. a plasmid origin of replication (ori);
2. a selectable marker, for example ampr;
3. a cos site, for re-circulating;
4. a suitable restriction site for cloning .
cos
ampr
5kb
B
ori
Packaging into  phage
Infection
B
B
(32- 47kb)
37-52 kb
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Bacteriophage M13
Genome features: Size is small (6.7 kb); Single-stranded; Circular
genome; DNA; Positive-sense.
Infection: M13 particles attach specifically to E.coli sex pili (encoded
by a plasmid called F factor), through a minor coat protein (g3p).
Binding of g3p induces a structural change in the major capsid
protein. This causes the whole particle to shorten, injecting the
viral DNA into the host cell.
g3p
g6p
end
g8p
RF
Host
enzymes
ini
g9p
g7p
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
E.coli/M13 phage vectors
Structure:  The phage particles contain a
6.7 kb circular ssDNA.  After infection
of a sensitive E. coli host, the
complementary strand is synthesized, like
a plasmid, and the DNA replicated as a
dsDNA, the replicative form (RF).
Features:  The host cells can continue to
grow slowly.
• ssDNA: The single-stranded forms are
continuously packaged and released from
the cells as new phage particles. ssDNA
has a number of applications, including 
DNA sequencing and  site-directed
mutagenesis.
• dsDNA: The RF (dsDNA) can be purified
in vitro and manipulated exactly like a
plasmid.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Cloning in M13
Purpose: When the single-stranded DNA of a fragment is
required, a M 13 vector can be used as a common cloning tool.
Preparation of ssDNA:
1. Cloning: standard plasmid cloning method can be used to
incorporate recombinant DNA into M13 vectors;
2. Transformation: the M13 then infects sensitive E. coli cells;
3. Plating: the host cells grow to form the plaques;
4. Isolation: the ssDNA may then be isolated from phage particles
in the growth medium of the plate.
Screening: Blue-white screening using MCSs and lacZ' has been
engineered into M13 vectors.
Examples: The M13mpl8 and M13mp19, which are a pair of
vectors in which the MCS are in opposite orientations relative
to the M13 origin of replication.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Hybrid plasmid-M13 vectors
Definition: A number of small plasmid vectors, for example
pBlue-script, have been developed to incorporate M13
functionality.
Structure: They contain both plasmid and M13 origins of
replication, but do not possess the genes required for the full
phage life cycle.
Working ways:
1. Plasmid way: they normally propagate as true plasmids, and
have the advantages of rapid growth and easy manipulation of
plasmid vectors;
2. Phage way: they can be induced to produce single-stranded
phage particles by co-infection with a fully functional helper
phage, which provides the gene products required for singlestrand production and packaging.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
H3 YAC and BAC
• Cloning large DNA fragments
• YAC vectors
• BAC vectors
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Cloning large DNA fragments
• Problems:
1. The analysis of genome organization and the identification of
genes, particularly in organisms with large genome sizes
(human DNA is 3  109 bp, for example) is difficult to use
plasmid and bacteriophage  vectors, since the relatively small
size capacity of these vectors for cloned DNA means that an
enormous number of clones would be required to represent the
whole genome in a DNA library.
2. In addition, the very large size of some eukaryotic genes, due to
their large intron sequences, means that an entire gene may not
fit on a single cloned fragment.
• Solution: Vectors with much larger size capacity have been
developed to solve these problems.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Yeast/YAC vectors
SnaBI
CEN4 is the centromere of chromosome 4 of
Yeast. The centromere will segregate the
daughter chromosomes.
ARS is autonomously replicating sequence,
its function is as a yeast origin of
replication.
TRP1 and URA3 are yeast selectable markers,
one for each end, to ensure the right
reconstituted YACs survive in the yeast
cells.
TEL is the telomeric DNA sequence, which is
extended by the telomerase enzyme inside
the yeast cell.
SUP4 is a gene, which is insertionally
inactivated, for a red-white color test, like
blue-white screening in E. coli.
S
pYAC3
B
B
BamHI
Function: YAC vectors can accept genomic DNA fragments of more
than 1 Mb, and hence can be used to clone entire human genes.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
E.coli/BAC vectors
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
H4 Other Eukaryotic Vectors
•
•
•
•
•
•
•
Cloning in eukaryotes
Transfection of eukaryotic cells
Shuttle vectors
Yeast/episomal plasmids
Agrobacterium tumefaciens/Ti plasmid
Insect cell/Baculovirus
Mammalian cell/viral vectors
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Cloning in eukaryotes
Reasons:
• E. coli as host: Many eukaryotic genes and their
control sequences have been isolated and analyzed
using gene cloning techniques based on E. coli as host.
• Eukaryotic Vectors: However, many applications of
genetic engineering (see Section J) require vectors for
the expression of foreign genes in different eukaryotic
species, for example:
1. Large-scale production of eukaryotic proteins;
2. Engineering of new plants;
3. Gene therapy for human.
• Such kinds of vectors designed for a variety of hosts
are discussed in this topic.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Transfection of eukaryotic cells
Problem: The transfection of DNA into eukaryotic cells is
more problematic than E.coli transformation, and
efficiency of the process is much lower.
Reasons and solutions:
• In yeast and plant cells, the cell wall must be digested,
which may then take up DNA easily.
• Animal cells in culture take up DNA at low efficiency.
If it is treated on their surface with calcium phosphate,
the efficiency may be increased.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Transfection of eukaryotic cells
Other transfection techniques:
• Electro-poration: By treatment of
the cells with a high voltage,
which opens pores in the cell
membrane.
• Micro-injection: foreign DNA
may be microinjected into cells,
by using very fine glass pipettes.
• Micro-projectiles: DNA may be
introduced by micro-projectiles
which fire metallic coated with
DNA at the target cells.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Shuttle vectors
Definition: They are the vectors that can
shuttle between more than one host, for
example, one is E. coli and the other is
yeast.
Structure and function: Most of the vectors
for use in eukaryotic cells are constructed
as shuttle vectors.
• In E. coli:
– This means that they can survive and
have the genes (ori and ampr ) required E.coli
for replication and selection in E. coli.
• In the desired eukaryotic cells:
Yeast
– They can also survive in the desired
host cells, and let the target insert
sequences take effects.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Yeast episomal plasmids
Structure of YEps
a ori: for replication in E.coli
a ampr: for selection in E. coli
a 2 origin: for replication in
yest
LEU2: is homologous gene
and a selectable marker in
yeast, involved in leucine
synthesis.
X gene: a shuttle sequence.
Function of YEps
• It replicates as plasmids
• It integrates into a yeast
chromosome by homologous
recombination.
Section H: Cloning Vectors
ori
ampr
YEps
2 origin
X gene
LEU2
Yang Xu, College of Life Sciences
Agrobacterium tumefaciens/Ti plasmid-I
Definition: Ti plasmid is a kind of plasmid which commonly used
to transfer foreign genes into a number of plant species.
plant DNA
T-DNA
Ti
expression
Function: The bacterium A. tumefaciens can infects and transfer
foreign genes into:
1. Dicot plants: tomato, tobacco;
2. Monocot plants, for example rice.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Improving: Disarmed T-DNA
shuttle vectors
– The recombinant T-DNA can be
constructed in a E. coli plasmid;
– Then transform into the A.
tumefaciens cell carrying a
modified Ti plasmid without TDNA.
– Infecting plant cell culture with
A. Tumefaciens.
– Plating transformed clones.
– Regenerate plant using hormone
In E.coli
Ti plasmid
Modified
Ti plasmid
transform
Infection
In A. tumefaciens
Plating
Advantage:
Integrate cloned genes easily, and
The recombinant plants can be
reconstituted from the
transformed cells.
Section H: Cloning Vectors
Regeneration
Yang Xu, College of Life Sciences
Insect cell/Baculovirus
Definition: Baculovirus is an  insect virus
which can be used for the overexpression of
 animal proteins  in insect cell culture.
Mechanism:
• Viral promoter: This viral gene has an
extremely active promoter.
• Insect cell culture: The same promoter can be
used to drive the over-expression of a foreign
gene engineered into the baculovirus genome.
Function: This method is being used increasingly
for large-scale culture of proteins of animal
origin, since the insect cells can produce
many of the post-translational modifications
of animal proteins, which a bacterial
Baculovirus-infected SF21 cells
expression system cannot.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Mammalian cell/viral vectors
• SV40: This virus can
infect a number of
mammalian species. The
SV40 genome is only
5.2 kb in size.
• Since it has packaging
constraints similar to
phage , so it can be not
used for transferring
large fragments.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
Mammalian cell/viral vectors
• Retroviruses: They have a
ssRNA genome, which is copied
into dsDNA after infection. The
DNA is then stably integrated
into the host genome by a
transposition mechanism. They
have some strong promoters,
and they have been considered
as vectors for gene therapy (see
Topic J6), since the foreign
DNA will be incorporated into
the host genome in a stable
manner.
Section H: Cloning Vectors
Yang Xu, College of Life Sciences
That’s all for Section H
Section H: Cloning Vectors
Yang Xu, College of Life Sciences