CLONING VECTORS:

TYPES AND CHARACTERISTICS

 VECTOR
•
S
A cloning vector is a DNA molecule in which foreign DNA can be
inserted or integrated and which is further capable of replicating within
host cell to produce multiple clones of recombinant DNA.

 Small DNA molecule capable of self replication.

 Cloning vehicle or Cloning DNA.
 Carrier of DNA fragment.
 E.g. Plasmid, Phage, Hybrid vector, Artificial
Chromosomes.
 CHARACTERISTICS
 Self replication, multiple copies.
 Replication origin site.
 Cloning site.
 Selectable marker gene.
 Small size.
 Low molecular weight.
 Easily isolated & purified.
 Easily isolated into host cell.
 Control elements – promoter, operator, ribosome binding site.
Two types :-
TYPES
1) Cloning Vectors
Propagation or cloning of DNA insert inside a suitable host cells.
Examples: Plasmids, Phage or Virus
Obtaining millions of copies. Uses :-
Genomic library. Preparing
probes.
Genetic Engineering
Experiments.
 Selection of cloning vector
depends on :-
(a) Objective of cloning
experiment
(b) Ease of working.
2) Expression Vectors

 Express the DNA insert producing specific protein.

 They have prokaryotic promoter.
 Ribosome binding site.
 Origin of replication.
 Antibiotic resistance gene.
 Expression vectors with strong promoters.
 Inducible Expression Vectors.
 Eukaryotic expression vectors.
 AGENTS USED AS VECTORS
 PLASMIDS
BACTERIOPHAGES
COSMID
ARTIFICIAL CHROMOSOME VECTORS

In 1973, Cohen described first

successful construction of recombinant vector.
Plasmid PSC101 - Ecoli
 VECTO TARGET HOST CELL
R
• Plasmid Bacteria, Streptomyces
• Bacteriophages Bacteria
• Cosmid Bacteria
• Yeast Cloning Vectors Yeasts
• Ti & Ri Plasmids Transformation of
cloned gene in higher
plants.
 PLASMID
 Extra chromosomal DNA molecules.
 Self replicating.
 Double stranded.
 Short sequence of DNA.
 Circular DNA molecules.
 Found in prokaryotes.
CHARACTERISTICS
a. Minimum amount of DNA.
b. Two suitable markers for
identification .
c. Single restriction site.
d. More restriction enzyme.
e. Size range 1kb – 200kb.
f. Relaxed replication control.
NATURAL AND ARTIFICIAL PLAMIDS

• Plasmids isolated from bacteria & directly used for

gene cloning without any modification :-Natural
Plasmid
• But not used in gene cloning due to large size,confer
pathogenicity etc
• Artificial/derived plasmids constructed by cutting out
unwanted portions from wild type plasmids.Eg:pBR322
• These are of much use in gene transfer
EXAMPLES OF PLASMID VECTORS
 pBR322
 pBR327
 pBR325
 pBR328
 pUC8
 pUC9
 pUC12
 pUC13
 pGEM3Z
 PBR322
 Cloning Vector.
 15 copies.
 Reconstructed plasmid.
 Derived from Ecoli plasmid- ColE1.
 PBR322 – 4362 base pairs
P – denotes Plasmid B – Scientist
Boliver R - Rodriguez
322 – number given to distinguish.
 Ampilicin resistance gene derived from
RSF2124.
 Tetracycline resistance gene from
PSC101.
Structure of E.Coli plasmid cloning vector pBR322
 Screening
• Screening for pBR322 re­combinants is performed in the following way.
After transformation the cells are plated onto ampicillin medium and
incubated until colo­nies appear.
• All of these colonies are trans-formants (remember, untransformed cells are
amps and so do not produce colonies on the selective medium), but only a
few con­tain recombinant pBR322 molecules: most con­tain the normal, self-
ligated plasmid.
• To iden­tify the recombinants the colonies are replica plated onto agar
medium that contains tetra­cycline.
• After incubation, some of the original colonies regrow, but others do not.
• Those that do grow consist of cells that carry the normal pBR322 with no
inserted DNA and, therefore, a functional tet­racycline resistance gene
cluster (ampR tetR).
• The colonies that do not grow on tetracycline agar are recombinants
(ampR tefs); once their positions are known, samples for further study can
be recovered from the original ampicillin agar plate.
Advantages of pBR322:

1. Small size (~ 4.4 kb) enables easy purifi­cation and manipulation.

2. Two selectable markers (amp and tet) al­low easy selection of recombinant
DNA.
3. It can be amplified up to 1000-3000 copies per cell when protein synthesis
is blocked by the application of chloramphenicol.
Disadvantages of pBR322:
1. It has very high mobility i. e; it can move to another cell in the presence of a
conjugative plasmid like F-factor. The nic-bom (bom=basis of mobility) region of
pBR322 is responsible for this feature. Due to this, the vector may get lost in a
population of mixed host cells.

2. There is a limitation in the size of the gene of interest that it can accommodate.

3. Not a very high copy number is present as is expected from a good vector.

4. Although insertional inactivation of an antibiotic resistance gene provides an ef­

fective means of recombinant identifica­tion, the method is made inconvenient by
the need to carry out two screenings, one with the antibiotic that selects for trans-
formants, followed by the second screen, after replica plating, with the an­tibiotic
which distinguishes recombinants.

This makes the screening process time- consuming and laborious.

Another vector pBR327 was derived from pBR322, by deletion of nucleotides

between 1,427 to 2,516. These nucleotides are deleted to reduce the size of the vector
and to elimi­nate sequences that were known to interfere with the expression of
cloned DNA in eukaryotic cells. pBR327 still contains genes for re­sistance against
two antibiotics (tetracycline and ampicillin).
 PUC Vectors
 Popular Ecoli cloning vector.
 Derivative of pBR322.
 Ampicillin resistance gene.
 ColEI – origin of replication.
 2700 base pairs (smaller than pBR322 ).
 high copy number (500 to 600 copies of
the plasmid per cell )
 lac Z gene derived from Ecoli.
 Polylinker sequence having unique restriction sites
lies in lac region.
The Nomenclature of pUC Vectors:
The nomenclature of ‘pUC’ can be understood with the
following explanation:
1. ‘p’ indicates the plasmid.
2. ‘UC’ stands for university of California where it was first
developed by J. Mess­ing et al.

We also see many numbers after this like pUC8, pUC18,

pUC19 and so on. They are just the series of pUC and have
been named just to separate from each other.
The construction of pUC vectors:
1. Origin of Replication: It is derived from the origin of replication of pBR322.The
ColE1origin of replication of pBR322 has been modified by carrying out a chance
mutation so that each transformed E. coli cell has 500-600 copies of the plasmid.

2. Selectable Marker: It has an ampicil­lin resistant gene. The transformed host cells
can grow on media having ampicillin whereas non-transformed cells die.

3. lac Z’ gene having MCS. The lac Z’ is incorporated into this vector codes for the
enzyme beta-galactosidase which acts on a chromogenic substrate called X-gal
(present in bacterial culture media). The expression of lac Z’ gene is in­duced by
another compound present in the same media called Iso-propyl-thiogalactoside
(IPTG).

When the enzyme substrate reaction takes place, then the X- gal is converted from white to a blue com­
pound. Now the lac Z’ gene itself has MCS. Hence, when the gene of interest has been introduced into
the lac Z’ gene, then it fails to code for beta-galactosidase and thus in this case the substrate (X-gal) is
never converted to any other colour. This type of screening is called blue-white screen­ing.
BLUE WHITE SCREENING
Uses of pUC Vectors:
pUC vectors can be used both as cloning vec­tor and
expression vector. When used as an expression vector
its sequences are slightly modified to meet necessary
requirements.
Advantages of pUC Vectors:
The pUC vectors offer following major advan­tages
over pBR322 vectors:
(a) High copy number of 500-600 copies per cell.
(b) Easy and single step selection.
(c) The unique restriction sites used for clon­ing are
clustered within the MCS. This allows cloning of a DNA
fragment having two different sticky ends.

Disadvantages of pUC:
It cannot accommodate a gene of interest larger than
15kb.
 PHAGE
 Cloning large DNA fragments.
 Linear Phage molecule.
 Efficient than plasmid.
 Used in storage of recombinant
DNA.
 Commonly used Ecoli phages :-
λ phage
M13 Phage
Phage M13 Vectors
Filamentous bacteriophage of Ecoli.
Used for obtaining single stranded copies.
DNA sequencing.
Single stranded.
Inside host cell become double stranded.
 HYBRID VECTOR
 Component from both plasmid & phage chromosomes.
 Helper phage provided.
 Developed in 1978 by Barbara Hohn & John Collins.
 30 – 40 Kb
 Origin of replication, cloning site, marker gene, DNA cos site.
 Smaller than plasmid.
 Use – construction of genomic libraries of eukaryotes.
 e.g. Cosmid, Phagemid
 COSMIDS
• Combine parts of the lambda chromosome with parts of plasmids.
• Behave both as plasmids and as phages.
• Contain the cos sites of λ phage and plasmid
origin of replication.
• The presence of the cohesive end site, cos λ in a plasmid
allows the plasmid to be packaged in vivo into viral
particles. But, cosmids do not have genes that encode
viral proteins. Therefore, the formation of viral particles
is prohibited in cosmids.
• cosmids can accommodate up to 45 kb sized fragments.
Since cosmid vectors are capable of inserting large DNA
fragments when compared to plasmids, they are suitable
for cloning large mammalian genes or multi-gene
fragments.
Structure of Cosmid
• Origin of replication (ori).
• Restriction sites for cleavage and insertion of foreign DNA.
• Selectable marker from plasmid.
• A cos site - a sequence yield cohesive end (12 bases). Ampicillin
resistance gene (amp).

1. An example of a commonly used cosmid is pHV79 which is

nothing but pBR322 containing the cohesive end site cos λ
and which can accommodate up to 45 kb sized inserts.
2. Next example is pJB8
The following are the steps for construction of a cosmid
library:

(i) Cleavage of the genome by partial digestion with restriction

endonuclease,
(ii) Sizing of the fragments by gel electrophoresis or velocity
centrifugation;
(iv) Ligation of the genomic DNA and the cosmid DNA;
(v) Packaging the ligated DNA into infectious phage particles;
(vi) Transduction into E. coli.
Therefore, it is evident that different vectors have different capacity of
carry foreign DNA
A great advantage of such a cosmid vector is that:

(1) Gene libraries consisting of a smaller number of clone members can span
the whole genome of an organism. For example, the genome of Escherichia coli
can be accommodated in just 120 cosmids.
(2) Other advantages are that large gene can be studied intact and genetic
linkage studies can be carried out at the molecular level.
(3) An important practical advantage of a cosmid is that background molecules
which do not have the intact and genetics linkage studies can be carried out at
the molecular level.
(4) An important practical advantage of a cosmid is that background molecules
which do not have inserts or have smaller inserts are eliminated during
packaging. This is not possible to achieve with plasmid cloning vectors.
(5) Besides, the frequency of transformation of the lambda capsids with an in
vitro packaging extract is much higher than the transformation frequency of
plasmids.
 PHAGEMIDS
A phagemid is a plasmid that contains an f1 origin of replication from an f1 phage.
It can be used as a type of cloning vector in combination with filamentous phage M13.
A phagemid can be replicated as a plasmid, and also be packaged as single
stranded DNA in viral particles.
Phagemids contain an origin of replication (ori) for double stranded replication, as well
as an f1 ori to enable single stranded replication and packaging into phage particles. 
Many commonly used plasmids contain an f1 ori and are thus phagemids.
 PHAGEMIDS
1. A phagemids is a hybrid of a plasmid and a filamentous coliphage that can be
propagated in either form.
2. The coliphage could be either of the three virtually identical phages, M13, fd or f1.
3. These are male specific phages (carrying F pilus) that contain single stranded
circular DNA as their genome.
4. Upon infection of E. coli by the bacteriophage, double stranded DNA is first formed
as the replicative intermediate.
5. Finally single stranded DNA is packaged into the virion.
6. Both the replication origins of the plasmid and the coliphage are incorporated in
the phagemid.
7. The auxiliary replication functions necessary in trans for the coliphage replication
are not, however, incorporate in the phagemid.
8. Hence, replication from the coliphage origin can take place only in the presence of
a helper phage.
9. Although M13 vectors are very useful for the production of single-
stranded versions of cloned genes they do have one disadvantage.
10. There is a limit to the size of DNA fragment that can be cloned with an
M13 vector, with 1500bp generally being looked on as the maxi­mum
capacity.
11. To get around this problem a number of novel vectors have been
constructed which are the hybrids of plasmids and M13 vectors.
12. We call them phagemids (‘phage’ from M13 bacteriophage and ‘mid’
from plasmid).
Construction of Phagemid Vector:

A typical phagemid has following parts:

1. Phage M13/F1 origin of replication (M13 and F1 phages closely
resemble to each other).
2. A portion of lac Z’ gene driven by lac pro­moter.
3. A multiple cloning site (MCS) with lac Z’ gene.
4. Phage T7 and T3 promoter sequences flanking the MCS sequences.
5. ColE1 origin of replication.
6. ampR resistant gene.
Plasmids that carry the M13 replication origin in addition to a
conventional origin of dsDNA synthesis can be replicated either as
dsDNA from the latter or as single-stranded DNA from the M13 origin.
Replication from the M13 origin requires the appropriate pro­teins (such
as gene II protein) to be provided from a helper phage also replicating
within the cell.
Replication generates single-stranded DNA which can then be packaged
into phage coats. Examples of phagemids are the vectors pUC118, 119 and
120.
They are replicated as plasmids until the cell containing them is co-
infected with a helper phage, such as M13K07, which provides the
proteins for single-stranded DNA synthesis and packaging. M13K07 is an
M13 phage that has been modified, most im­portantly by the
incorporation of a plasmid replication origin.
Replication from this origin allows the helper phage to be present in a
high copy number per cell and, therefore, to pro­vide the larger quantities
of the proteins that are required to replicate and package the phagemid
molecule.
Uses of Phagemid Vectors:

This vector is a multipurpose vector as it can serve as

following:
1. A cloning vector.
2. An expression vector
3. A sequencing vector.

Advantages of Phagemid Vectors:

The main advantage of the phagemid system is that it can be
used to provide single-or double-stranded material without any
re-cloning.