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.
8. Promoter region for expression vector
Two types:
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.ƛEg. 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 process that can
Several different kinds of plasmid may be found in a single cell, including
result in a conjugative plasmid spreading from one cell to all the more than one different conjugative plasmid at any one time. In fact, cells of
other cells in a bacterial culture. E. coli have been known to contain up to seven different plasmids at once.
To be able to coexist in the same cell, different plasmids must be
Conjugation and plasmid transfer are controlled by a set of compatible . If two plasmids are incompatible then
one or the other will be rapidly lost from the cell.
transfer or tra genes, which are present on conjugative plasmids
but absent from the non-conjugative type.
 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

Tet R
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
pBR322 replicated in [Link], of a component of beta- galactosidase
p ------- plasmid gene
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 plasmids (R1, R6-5, and pMB1).
Repressor of primer (Rop ) is a small dimeric protein that participates in the mechanism
thatƛcontrolsƛthe copy number of plasmid of the ColE1 family by increasing the affinity
between two complementary RNAs. ColE1ƛisƛaƛplasmidƛfoundƛinƛbacteria.ƛ
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 cells with high efficiency, the cloned Bacteriophages
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 ƛƛ 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 ƛƛ 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 Eco RI site located in the c I 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 ′ƛgene carried by the
vector

ƛ EMBL4 an carry up to 20 kb of new DNA

 Phage vector Properties

Replacement Vectors?
Replacement vector orƛsubstitution vector ƛisƛ a type of phage vector developed from the
removal of a middle ‘fillerƛfragment’ƛregion of phage DNA. The desired foreign DNA insert
replaces theƛphageƛDNA.

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
restriction site introduced
the removal of a middle ‘filler
within the phage genome at the
fragment’ƛregion of phage DNA
site of optional DNA.
Size of Insert Fragments
05-11 kb length 08-24 kb length
Filler Fragment
Filler fragment gets replaced by
No filler fragment
a foreign insert
ƛƛFunction
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
A unique cleavage site is
that are not essential for the
present
lytic cycle
 M13—aƛfilamentousƛphage
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 ƛƛ 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