Vectors
a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell for the purpose of
cloning and expression. Cloning vectors are used to clone target DNA whereas expression vectors are engineered so
that desirable target DNA can be transcribed in RNA and translated in to protein.
A viral DNA or plasmid is generally used as a vector.
Important features of a cloning vector are as follows:
1. Ability to replicate in host cells: must have origin of replication for autonomous replication within the host cell.
2. Unique restriction sites for insertional cloning: All cloning vectors have features that allows a target DNA to be
conveniently inserted into the vector. This may be a multiple cloning site (polylinker site) which contains many
unique restriction sites
3. Genetic marker to select for host cells containing the vector.
1) Selectable marker: encodes a product that allows the growth of one type of cells under specific conditions
that kill or restrict the growth of other types of cells. (antibiotic resistance gene such as Amp r
2) Screenable marker: also called reporter gene, gives a product that can be detected using a simple
quantitative assay.
4. Low molecular weight:
Advantages: the Plasmid is more resistant to damage by shearing and is readily isolated from host cells
These plasmids are present in multiple copies
Less chance of having multiple restriction site on the vector for RE
5. It should possess multiple cloning sites.
6. It should be small in size so that it can easily integrate into the host cell.
7. It should be capable of inserting a large segment of DNA.
Two types:
8. Promoter region for expression vector
Cloning and Expression vector An expression vector, otherwise known as an expression construct, is
usually a plasmid or virus designed for gene expression in cells.
A vector is a DNA molecule that is used to carry a foreign
DNA into the host cell. It has the ability to self replicate
and integrate into the host cell. Eg. Plasmids,
bacteriophages, Phagemids, BAC, YAC
Use of antibiotic resistance as a selectable marker or a plasmid
Replication strategies for non integrative plasmid
and episome
Size and copy number
The copy number refers to the number of
molecules of an individual plasmid that are
normally found in a single bacterial cell.
Some plasmids, especially the larger ones, are
stringent and have a low copy number of perhaps
just one or two per cell; others, called relaxed
plasmids, are present in multiple copies of 50 or
more per cell.
Conjugation and compatibility
Plasmids fall into two groups: conjugative and non-
conjugative
Conjugative plasmids are characterized by the ability to
promote sexual conjugation between bacterial cells, a
Several different kinds of plasmid may be found in a single cell,
process that can result in a conjugative plasmid including more than one different conjugative plasmid at any
spreading from one cell to all the other cells in a one time. In fact, cells of E. coli have been known to contain up
to seven different plasmids at once. To be able to coexist in the
bacterial culture.
Conjugation and plasmid transfer are controlled by a same cell, different plasmids must be compatible. If two
plasmids are incompatible then
set of transfer or tra genes, which are present on one or the other will be rapidly lost from the cell.
conjugative plasmids but absent from the non-
Plasmid classification
• Fertility or F plasmids carry only tra genes and have no characteristic beyond the ability to promote conjugal
transfer of plasmids. A well-known example is the F plasmid of E. coli.
• Resistance or R plasmids carry genes conferring on the host bacterium resistance to one or more antibacterial
agents, such as chloramphenicol, ampicillin, and mercury. R plasmids are very important in clinical microbiology as
their spread through natural populations can have profound consequences in the treatment of bacterial infections. An
example is RP4, which is commonly found in Pseudomonas, but also occurs in many other bacteria.
• Col plasmids code for colicins, proteins that kill other bacteria. An example is ColE1 of E. coli.
• Degradative plasmids allow the host bacterium to metabolize unusual molecules such as toluene and salicylic acid,
an example being TOL of Pseudomonas putida.
• Virulence plasmids confer pathogenicity on the host bacterium; these include the Ti plasmids of Agrobacterium
tumefaciens, which induce crown gall disease on dicotyledonous plants.
Plasmid vector
TetR
pUC produced as university of California
Contains a polylinker and unique cloning
sites for multiple restriction nucleases,
ampicillin resistance
In addition, a selection of recombinants
is achieved by insertional inactivation of
a component of beta- galactosidase gene
pBR322 replicated in [Link],
p ------- plasmid
BR------- Boliver and Rodriguez
322----- distinguishes it from other plasmid developed in the same lab
Relatively high copy number 20-30 copies per cell
Genetically engineered from DNA derived from 3 different naturally occurring
Repressor of primer (Rop) is a small dimeric protein that participates in the
mechanism that controls the copy number of plasmid of the ColE1 family by
increasing the affinity between two complementary RNAs. ColE1 is a plasmid found in bacteria.
Not a very high copy number,
but 3rd point is saying 1000-3000
copies how?
It is possible only when the
protein synthesis is blocked by
chloramphenicol
Cloning vectors based on viral DNA
Viral vectors are those in which the gene or genes of interest are
incorporated into the genomes of a virus. Because viruses infect cellsBacteriophages
with high efficiency, the cloned gene can be introduced into cells at a
significantly higher frequency than by simple transformation.
Cloning vectors based on Lambda phage
Lambda phage, a temperate phage, infects [Link] and replicates by a lytic or lysogenic pathway.
Genome consist of single linear dsDNA of ~49kb. However, at either end of the molecule is a
short 12 nucleotides stretch, in which the DNA is ss described as cohesive or sticky ends. These
are called cos ends, play 2 role during infection cycle.
1st allow liner DNA molecule that is injected to circularize
2nd role come in to picture during lytic cycle. During lytic cycle a large number of new lambda
DNA molecules are produced by the rolling circle mechanism of replication. The result is a
catenate consisting of a series of linear lambda genomes joined together at the cos sites. Now
these cos sites acts as recognition sequences for endonucleases that cleaves the catenate at
the cos sites, producing individual lambda genomes.
The phage infection cycle
The general pattern of infection, which is the same for all types of
phage, is a three-step process:
1 The phage particle attaches to the outside of the bacterium and
injects its DNA chromosome into the cell.
2 The phage DNA molecule is replicated, usually by specific phage
enzymes coded by genes in the phage chromosome.
3 Other phage genes direct synthesis of the protein components of the
capsid, and new phage particles are assembled and released from the
bacterium.
With some phage types the entire infection cycle is completed
very quickly, possibly in less than 20 minutes. This type of rapid
infection is called a lytic cycle, as release of the new phage
particles is associated with lysis of the bacterial cell. The
characteristic feature of a lytic infection cycle is that phage DNA
replication is immediately followed by synthesis of capsid
proteins, and the phage DNA molecule is never maintained in a
stable condition in the host cell.
The general pattern of infection of a bacterial cell by a
bacteriophage
lytic cycle
Lysogenic phages
In contrast to a lytic cycle, lysogenic infection is characterized by
retention of the phage DNA molecule in the host bacterium,
possibly for many thousands of cell divisions. With many lysogenic
phages the phage DNA is inserted into the bacterial genome, in a
manner similar to episomal insertion
The integrated form of the phage DNA (called the prophage) is
quiescent, and a bacterium (referred to as a lysogen) that
carries a prophage is usually physiologically indistinguishable
from an uninfected cell. However, the prophage is eventually
released from the host genome and the phage reverts to the lytic
mode and lyses the cell.
The lysogenic infection cycle of bacteriophage lambda
There is one problem with the lambda phage DNA as cloning vector. SIZE LIMITATION. Normal lambda phase genome size is ~49kb, whereas the
capacity of capsid to incorporate maximum genome size is about 53kb. Thus a ƛ phage DNA molecule can be increased in size by addition of only 3kb of
new DNA. However, this problem has overcome by deletion of non-essential DNA of about 15kb. This means that as much as 18kb of new DNA can now
be added. The non-essesntial region, in fact contains the genes involved in lysogenic pathway. A deleted ƛ genome is therefore non-lysogenic and can
follow only lytic cycle.
insertion vector is the simplest form of lambda cloning vectors. In fact, it is a type of phage vector
that has a unique restriction site introduced within the vector genome at the position of optional
DNA. Furthermore, the phage DNA remains without a removal. This non-removal of the phage DNA
curtails the size of the inserts (foreign DNA) to be cloned within the vector. Moreover, these vectors
are useful in cDNA cloning and expression. GT10, GT11, and Zap are examples of this vector.
Insertion vector consists of a single recognition site. The primary function of this vector is to
develop cDNA libraries derived from eukaryotic mRNA sequences. Moreover, it can only
accommodate lengths of foreign DNA ranging in between 05-11 kb. Also, it possesses a unique
cleavage site for the insertion of foreign DNA.
Egt10 (Figure 6.12b), which can carry up to 8 kb of new DNA,
inserted into a unique EcoRI site located in the cI gene.
EZAPII (Figure 6.12c), with which insertion of up to 10 kb DNA
into any of 6 restriction sites within a polylinker inactivates the
lacZ′ gene carried by the vector
ƛEMBL4 an carry up to 20 kb of new DNA
Phage vector Properties
Replacement Vectors?
Replacement vector or substitution vector is a type of phage vector
developed from the removal of a middle ‘filler fragment’ region of phage
DNA. The desired foreign DNA insert replaces the phage DNA.
Replacement vectors are important in creating genomic libraries such as
EMBL4 and Charon40. These vectors can accommodate a larger length of
foreign DNA ranging between 08-24kb lengths. The filler region also consists
of a gene that makes the phage vector non-viable inside a bacterial host.
What are the Similarities Between Insertion and
Replacement Vectors?
•Insertion and replacement vectors are phage vectors.
•Both vectors accommodate foreign DNA inserts.
•They both are helpful in creating DNA libraries.
Difference Between Insertion and
Replacement Vectors?
Insertion vs Replacement Vectors
Insertion vector is a type of
Replacement vector is a type of
phage vector that has a
phage vector developed from the
restriction site introduced within
removal of a middle ‘filler
the phage genome at the site of
fragment’ region of phage DNA
optional DNA.
Size of Insert Fragments
05-11 kb length 08-24 kb length
Filler Fragment
Filler fragment gets replaced by a
No filler fragment
foreign insert
Function
Important to create cDNA Important in creating genome
libraries libraries
Examples
GT10, GT11, and Zap are EMBL4 and Charon40 are
examples examples
Cleavage Site
Cleavage site contains genes that
A unique cleavage site is present are not essential for the lytic
cycle
M13—a filamentous phage
M13 is an example of a filamentous phage and is completely
different in structure from ƛ. Furthermore, the M13 DNA molecule is
much smaller than the genome, being only 6407 nucleotides in
length. It is circular and is unusual in that it consists entirely of
single-stranded DNA.
The smaller size of the M13 DNA molecule means that it has room for
fewer genes than the ƛ genome. This is possible because the M13
capsid is constructed from multiple copies of just three proteins
(requiring only three genes), In addition, M13 follows a simpler
infection cycle than ƛ, and does not need genes for insertion into the
host genome.
Injection of an M13 DNA molecule into an E. coli cell occurs via
the pilus, the structure that connects two cells during sexual
conjugation. Once inside the cell the single-stranded molecule
acts as the template for synthesis of a complementary strand,
resulting in normal double-stranded DNA
This molecule is not inserted into the bacterial genome, but
instead replicates until over 100 copies are present in the cell.
When the bacterium divides, each daughter cell receives copies
of the phage genome, which continues to replicate, thereby
maintaining its overall numbers per cell.
new phage particles are continuously assembled and released,
about 1000 new phages being produced during each generation
of an infected cell.
Several features of M13 make this phage attractive as a cloning
vector.
1. The genome is less than 10 kb in size, well within the range
desirable for a potential vector
2. the double-stranded replicative form (RF) of the M13 genome
behaves very much like a plasmid, and can be treated as
such for experimental purposes
3. It is easily prepared from a culture of infected E. coli cells and
can be reintroduced by transfection
4. genes cloned with an M13-based vector can be obtained in
the form of single-stranded DNA. Single-stranded versions of
cloned genes are useful for several techniques, notably DNA
sequencing and in vitro mutagenesis.
5. M13 vectors are also used in phage display, a technique for
identifying pairs of genes whose protein products interact
with one another