VECTOR

A vector is a substance, usually a piece of DNA that carries a sequence of DNA

or other genetic material and introduces it into a new cell.
Vectors act as vehicles to transfer genetic material from one cell to the other for different
purposes like multiplying, expressing, or isolation. The DNA insert that is transmitted by a
vector is termed recombinant DNA, and the process is known as recombinant DNA
technology. Vectors usually have an insert, also known as a transgene, that carries the
recombinant DNA and a larger sequence called the backbone of the vector responsible
for the structure of the vector. Vectors have particular features that carry the gene
sequences and enable them to survive within the host cell. The process of gene transfer
also differs in different vectors where some enter the host cell and get incorporated into
the host DNA, whereas the others just pass the genetic material into the host cell and
recover themselves. Viruses and other particles can also function as vectors in processes
like transduction. Vectors can be reused for multiple processes as these can be recovered
at the end of the process.

FEATURES OF VECTORS
1. Origin of Replication (ori)
The origin of replication is a DNA sequence recognized by host replication machinery,
ensuring the vector is copied during cell division. Origins determine both copy number
and compatibility. High-copy origins like ColE1 in pBR and pUC plasmids generate
hundreds of copies per cell, providing abundant DNA or protein product. Low-copy origins,
such as those in BACs, reduce metabolic burden and maintain stability of very large
inserts. Compatibility refers to the ability of multiple plasmids with different origins to
coexist in the same host.
2. Size of the Vector
An ideal vector should be small in size, allowing easy manipulation and efficient
incorporation into host cells. Smaller vectors can also accommodate larger DNA inserts
and are easier to isolate and purify.

3. Easy Isolation and Purification

Vectors should be simple to recover from host cells for reuse in further cloning or transfer
processes. Small and stable vectors are preferable for this purpose.

4. Selectable Marker Genes

Selectable markers help differentiate transformed cells (cells that have taken up the
vector) from non-transformed ones. They are usually antibiotic resistance genes or
metabolic genes that allow selective growth under specific conditions. Examples:
 Bacterial markers: bla (ampicillin), tetR (tetracycline),
 Plant markers: PAT/bar, hpt
 Yeast markers: URA3 (permits growth only in uracil-deficient media)

5. Reporter Gene or Screenable Marker

Reporter genes allow visual or measurable distinction between recombinant and non-
recombinant clones. They produce colorimetric, fluorescent, or luminescent signals when
expressed. Common reporter genes:
 lacZ → produces β-galactosidase (blue/white screening using X-gal)
 GFP → green fluorescent protein
 GUS (β-glucuronidase)
 antibiotic hygromycin B.
Transformed plant cells
survive and grow in the
 luciferase → produces bioluminescence presence of hygromycin,
Example (Blue-White Screening): while non-transformed cells
β-galactosidase is an enzyme encoded by the lacZ gene in
bacteria like E. coli. Its main function is to hydrolyze lactose into glucose and galactose,
allowing the cell to use lactose as an energy source. In molecular biology, it is commonly
used as a reporter enzyme to detect the activity of the lacZ gene in cloning experiments.
X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) is a synthetic substrate for β-
galactosidase. It is colorless, but when cleaved by β-galactosidase, it produces a blue-
colored product, making it useful in techniques like blue-white screening to visually
distinguish bacterial colonies that express or lack functional βgalactosidase.
 Plasmid without insert → lacZ intact → β-galactosidase produced → X-gal cleaved →
blue colonies
 Plasmid with insert → lacZ disrupted → no enzyme → X-gal not cleaved → white
colonies

6. Multiple Cloning Site (MCS) or Polylinker

The MCS is a short DNA segment containing several unique restriction enzyme sites.
 It allows insertion of foreign DNA using specific restriction enzymes.
 The presence of multiple sites provides flexibility for directional cloning.
 In many vectors, insertion into the MCS disrupts a reporter gene, aiding in
identification of recombinant clones.

7. Regulatory Sequences
Vectors used for gene expression contain additional regulatory elements for controlled
transcription and translation:
 Promoters: Can be constitutive or inducible; control transcription rate.
 Ribosome Binding Sites (RBS): Ensure efficient translation in prokaryotes.
 Terminators: Signal the end of transcription.
 Polyadenylation signals: Stabilize mRNA in eukaryotes.
 Fusion tags (e.g., His-tag, GST, FLAG): Facilitate protein purification and detection.
 Epitope sequences: Enable antibody recognition of expressed proteins. (An epitope is
the specific part of antigen that is recognized by the immune system)

8. Easy Introduction into Host Cells

Vectors should be capable of being introduced efficiently into host cells through
transformation, transduction, or conjugation. The efficiency of this process depends on
the host type, vector structure, and transformation method.

9. Integration Capability (for Gene Transfer Vectors)

For gene therapy or stable genetic modification, vectors should be able to integrate the
recombinant DNA into the host genome, ensuring stable inheritance through cell
divisions.

10. Vector Stability

The insertion of foreign DNA should not interfere with the replication or stability of
the vector. The vector should maintain its structure and function throughout multiple
replication cycles.

TYPES OF VECTORS
Vectors can be classified into different groups depending on the purpose of the process
and the type of particles used in the process. The following are the commonly studied
group of vectors that are used for different purposes:

1. Cloning Vectors
 Cloning vectors are vectors that are capable of replicating autonomously and thus are
used for the replication of the recombinant DNA within the host cell.
 Cloning vectors are responsible for the determination of which host cells are
appropriate for replicating a particular DNA segment.

a. Plasmid vector
b. Bacteriophage vector
A bacteriophage vector is a phage-based cloning vehicle that uses the genome of a
bacteriophage to carry, replicate, and express foreign DNA inside bacterial cells.
Bacteriophages or phages have higher transformation efficiencies which increase the
chances of recovering a clone containing the recombinant DNA segments.

λ phage, which infects E. coli, is a commonly used cloning vector. Its DNA is ~50 kb
long and has cos ends allowing it to circularize inside the host cell.
For cloning large DNA fragments (up to ~20 kb), nonessential λ DNA regions are
replaced with the desired insert. The recombinant DNA is packaged into phage particles
in vitro and used to infect E. coli. Inside the host, the recombinant phage undergoes lytic
growth, forming plaques—clear zones of lysed cells—from which cloned DNA can be
recovered.
Types of λ Vectors
1. Replacement Vectors: A portion of λ DNA is removed and replaced with foreign DNA.
Used for large inserts. Recombinant phages (with insert) are viable, while non-
recombinants (without insert) are inviable, aiding selection.
2. Insertion Vectors: The vector is cut at a single restriction site, and the insert is added
without removing λ DNA. Suitable for smaller inserts.

Filamentous phages, particularly those of the Ff class such as f1, fd, and M13, serve
as important cloning vectors in biotechnology. These phages infect E. coli cells and
possess a circular single-stranded DNA (ssDNA) genome produced through rolling circle
replication, enclosed in a long, filamentous protein coat. They are highly suitable as
vectors because they readily accept foreign DNA inserts and provide single-stranded DNA
in a form that is easy to isolate and manipulate. Among them, the M13 bacteriophage is
the most widely used vector.

After infecting the host cell, the single-stranded DNA of M13 is converted into a double-
stranded replicative form (RF), which serves as the template for cloning and replication of
foreign DNA. Unlike lytic phages such as lambda, M13 does not destroy the host cell
during replication; instead, recombinant phage particles are continuously released
through the cell membrane without causing lysis. Each phage particle packages the “+”
strand DNA, which corresponds in sequence to the mRNA, allowing the isolation of pure
single-stranded DNA. This property makes filamentous phages, especially M13,
particularly useful for applications requiring single-stranded DNA, such as DNA
sequencing, mutagenesis, and probe preparation. However, they are generally not used
as primary cloning vectors for large DNA fragments but rather for subcloning when a
single-stranded form of DNA is needed.
c. Cosmid
The first cosmid vector was described by Collins in 1978. These are medium sized, hybrid
cloning vectors that combine features of plasmids and bacteriophage λ. Ori, MCS
and selectable marker are obtained from the plasmid and only the cos site region is taken
from lambda phage. They are designed to carry larger DNA fragments than ordinary
plasmids can accommodate. Cosmids generally range in size from 400bp to 30 kb
(~5kb) and can incorporate up to ~45 kb of foreign DNA.
The cos seq can’t act properly if the bp number
between two cos ends is not equal to or near
equal to the bp no. of the viral DNA. Effective
packaging takes place only when a minimum
amount of DNA is present. Suppose the length
of the viral DNA was 50kb and length of cosmid
is 5kb, now the insert size must have to be ~45
kb (so that the length of recombinant cosmid
become 50kb) in order to preserve the
function of cos gene, and incorporate it
correctly into phage capsid.
A cosmid vector may have one or two cos sites
The cos sequence (~200 bp) is a specific
sequence derived from λ phage and contains
two main functional parts:
1. Cohesive 5’ ends: A cosN site where DNA is nicked at
each strand, 12 bp apart, by terminase. This causes
linearization of the circular cosmid with two "cohesive"
or "sticky ends" of 12bp 5’ overhangs. (The DNA must be
linear to fit into a phage head.)
2. Packaging signal – a recognition sequence for λ phage
packaging machinery, enabling the cosmid DNA to be
efficiently packaged into phage heads. This consists of
cosB and cosQ.

The cloning of a foreign DNA in cosmid vector involves the

following steps:
(1) ligation of the foreign DNA between two cos sites; (2)
making a concatemeric DNA; (3) in vitro packaging to
introduce the DNA into the phage head to form the matured
phage particle; and (4) introduction of the cloned DNA
into E. coli by transduction. After their entry into the host
cell, the cosmids are maintained as plasmids.
The use and production of cosmids have increased due to their high efficiency in cloning
large DNA fragments and their suitability for constructing genomic libraries. A well-known
example of a cosmid vector is pHC79, a cos-containing derivative of pBR322.

d. Phagemids
Phagemids are artificial hybrid cloning vectors derived from plasmid and filamentous
phage (like M13) features. They carry a plasmid origin of replication and a phage origin
(f1 origin), allowing them to replicate as plasmids in normal condition or produce single-
stranded DNA when co-infected with a helper phage. This makes them especially useful
for techniques like site-directed mutagenesis, DNA sequencing, and phage display. Ex.
pEMBL phagemids, pBLUESCRIPT phagemids.
Limitations of phagemids: limited insert size, require helper phage for packaging, lower
efficiency of phage particle production, and more complex handling compared to standard
plasmids.
e. Bacterial artificial chromosome (BAC)
Bacterial artificial chromosomes (BAC) were developed by Mel Simmons and coworkers in
the early 1990s and are based on the fertility factor (F factor) of Escherichia coli. The F
plasmid, a ~ 100 kb circular double stranded DNA, is present is an E. coli cell in only 1-2
copies. The synthetic BAC vectors, which are only ~7.5 kb double stranded DNA circles
contain the replication origin oriS and the gene repE of the F plasmid that are responsible
for initiation and proper orientation of replication of the BAC vector.
The parA and parB genes of the F plasmid ensure efficient segregation of the F factor into
the daughter E. coli cells after its replication are also incorporated in the BAC vector. The
BAC vectors also contain multiple cloning sites (mcs), a selectable marker in the form of
antibiotic resistance and colour based identification (lac Z complementation system) of
recombinants carrying inserts.
The naturally occurring F2 factors consist of up to 25% of the E. coli genome integrated
into the basic F factor and are very stable. This characteristic of the F factor contributes
to the ability BACs to accommodate very large amount of external DNA to the extent of
300kb.

The recombinant BACs have been found to exhibit a lower level of rearrangement and
chimerism of the cloned DNA sequence than exhibited by YACs. The cloning of DNA in
BACs is done as is done in a plasmid, by linearising the vector with a restriction
endonuclease, treating with phosphatase and then ligating with the DNA fragments to be
cloned. E. coli has to be transformed by electroporation because of the large size of the
recombinant BAC.

f. Human artificial chromosome (HAC)

Human artificial chromosomes are extrachromosomal DNA fragments that act as a new
chromosome within the human cell. The use of human artificial chromosomes has
increased with advances in genetic engineering as it helps overcome problems commonly
associated with traditional vector systems. HACs can exist as single copy episomes
without integration into the host chromosomes allowing long-term stable maintenance.
Besides, there is no upper limit in the size of the DNA insert to be incorporated into a HAC
as entire genomic units can be used to mimic the natural gene expression. In spite of
numerous advantages, HACs have only been used for studies related to the structure and
function of human kinetochores. Limitations associated with HACs are due to technical
difficulties during gene loading and ill-defined structures of the vectors.

g. Yeast artificial chromosome (YAC)

YAC is an artificial cloning vector based on the yeast Saccharomyces cerevisiae
chromosome structure. It is designed to clone very large fragments of DNA, typically up to
1000 kb/ 1 Mb in length. These consist of a mixture of functional units from different
organisms, but once the insert DNA is cloned, these can function as normally replicating
yeast chromosomes. YAC mimics a true eukaryotic chromosome, replicating and
segregating like one in yeast. Used in genome mapping, physical mapping of eukaryotic
chromosomes, and sequencing projects (e.g., human genome project).
 Components: A YAC contains all essential elements required for replication and
segregation in yeast:
1. ARS (Autonomously Replicating Sequence): Acts as the origin of replication.
2. CEN (Centromere): Ensures proper segregation during cell division.
3. TEL (Telomeres): Provide stability to linear DNA ends.
4. Selectable marker genes: Allow selection of transformants (e.g., URA3, TRP1).
5. Restriction sites: For insertion of foreign DNA.

There are some limitations with using YAC as vectors as these introduce a high degree of
chimerism and insert rearrangement. Since these are eukaryotic cells, these are difficult
to handle and have lower efficiencies as compared to bacterial artificial chromosomes.
Different YACs have been created over the years that are then used for different
purposes. One example: pYAC4

h. YEP (Yeast Episomal Plasmid)

YEP is a type of artificial yeast cloning vector derived from the 2μ (2-micron) plasmid
naturally found in Saccharomyces cerevisiae (yeast). It replicates episomally — that is,
independently of the yeast chromosome, but within the same nucleus. Features:
 Origin of replication: Contains the 2μ origin, allowing autonomous replication in yeast.
 Selectable marker: Includes yeast marker genes like URA3, LEU2, or TRP1 for selection in
auxotrophic strains.
 High copy number: Maintains 20–50 copies per cell, giving a high yield of cloned DNA.
 Bacterial origin of replication (ori): Allows replication in E. coli for easy plasmid
manipulation in bacteria.
 Shuttle vector nature: Functions both in E. coli and S. cerevisiae, enabling cloning in
bacteria and expression in yeast.
 Presence of MCS (multiple cloning site): For insertion of foreign genes.

Category:
 Artificial vector (engineered for lab use)
 Shuttle vector (works in both yeast and bacteria)
 Cloning and expression vector (used for gene cloning and sometimes for protein
expression in yeast)

2. Viral Vectors
Viral vectors are modified viruses used to deliver foreign genes into host cells. They are
made non-pathogenic and can efficiently introduce genetic material for cloning, gene
expression, or gene therapy.
Viral vectors are broadly classified as:
a. Animal Virus Vectors
These are derived from viruses that infect animal cells. They are used in animal cell
culture systems or gene therapy in animals/humans can also be called Gene Therapy
Vectors. Common Examples:
Virus Type Key Features
SV40 (Simian DNA Double-stranded circular DNA virus of monkeys. Used for
Virus 40) virus cloning and expression in mammalian cells. Accepts small DNA
inserts (~5 kb). SV40 promoter and origin of replication are
strong and used in expression vectors.
Adenovirus DNA Linear dsDNA virus. Can infect both dividing and non-dividing
virus cells. High gene expression level, used in gene therapy.
Retrovirus RNA Converts RNA → DNA via reverse transcription. Integrates into
virus host genome → stable expression. Used in gene therapy and
transgenic animal studies.
AAV (Adeno- ssDN Integrates into host genome at specific sites. Used in long-term
associated A gene therapy.
virus) virus

b. Plant Virus Vectors

These are used to introduce foreign genes into plant cells for transient expression or
transgenic plant development. Common Examples:
Virus Type Key Features
Cauliflower Mosaic DNA Double-stranded circular DNA virus. Infects dicot plants.
Virus (CaMV or virus Has a strong 35S promoter, widely used in plant
CMV) expression vectors. Limited insert size (~1.5 kb).
Tobacco Mosaic RNA ssRNA virus infecting tobacco and related plants. Used for
Virus (TMV) virus high-level transient expression. Can carry small foreign
genes.

3. Expression vector
 Expression vectors are vectors that enable the expression of cloned genes in order to
determine the successful cloning process.
 Usually, cloning vectors do not allow the expression of a cloned gene which is why the
use of expression vectors is required.
 The use of expression vectors facilitates the processing of introns in prokaryotes as
these are designed with restriction sites next to the regulatory region.
 The restriction sites on the vectors result in splicing of the cloned gene to permit the
expression of the gene under the regulatory system.
 The regulatory system in expression vectors consists of a promoter sequence, a
termination sequence, along a transcription termination sequence. It also contains
Ribosome Binding Site (RBS), Start and Stop codons, MCS, Operators or Enhancers.
 The use of expression vectors is essential to determine the success of a cloning
procedure and the efficiency of selective markers on the vectors.
 Expression vectors can be plasmid-based or viral-based that are introduced into the
host cells in order to code for particular mRNAs.
 The expression vectors are often used for the production of proteins that can then be
visualized by different methods depending on the complexity of the host cell.
 Expression vectors are of varying degrees of complexity depending on whether they
are to be used in prokaryotic or eukaryotic cells.

4. Shuttle vector
 Shuttle vectors are that carry origins of replication from two different hosts, which
enables them to ‘shuttle’ between the two hosts. (2 or more ori sites)
 These vectors contain DNA plasmids that can usually replicate in both mammalian
cells as well as bacterial cells. Shuttle vectors function as hybrid vectors containing
DNA sequences from bacterial plasmids and mammalian viruses.
 The vectors contain three functional DNA sequences involved in the cloning process; a
viral replication origin, a bacterial replication origin, and a drug resistance gene. The
presence of different replication sites and repair sequences enable the recovery and
maintenance of these vectors in bacterial cells.
 There are three different shuttle vectors depending on the type of replication system
utilized by the vectors: Transiently replicating shuttle vectors that need to be
recognized by large T antigen in order to replicate in human cells. Episomal shuttle
vectors work to establish cell lines that can replicate permanently in the form of
plasmid DNA containing the DNA insert. Integrated shuttle vector undergoes
replication only after fusion with particular cell types for gene expression.

5. Secretion vector
 Secretion vectors are a type of specialized expression vector that expresses the cloned
genes in order to produce proteins at locations other than the cytoplasm. The
transport of protein product from the cell is achieved by the fusion of the inset DNA
with a nucleotide sequence encoding the peptide of an easily secreted protein. The
use of secretion vector has many advantages like higher yield, simple purification
process, and improved protein stability. Secretion vectors can be designed for more
than one type of prokaryotes or eukaryotes, including mammals.
 A commonly associated problem with the incorporation of a protein of eukaryotic origin
into a prokaryotic host is the overexpression of the protein. This problem is solved by
the use of secretion vectors that alleviate the formation of inclusion bodies.
 Secretion vectors have replaced cloning vectors in processes focusing on the
production of proteins and the expression of eukaryotic DNA fragments.

VECTORS BASED ON FUNCTION AND CHARACTERISTICS (summary)

Type of Main Features Examples Purpose / Use
Vector

1. Cloning Used for replication and pBR322, pUC18, pUC19, To clone and
Vectors amplification of foreign DNA pBluescript maintain DNA
fragments; contain ori, fragments inside
selectable marker, and MCS. a host.

2. Contain regulatory pET series, pGEX, pMAL To express foreign

Expression sequences (promoter, genes and
Vectors terminator, RBS, operator) for produce proteins.
transcription and
translation of the inserted
gene into protein.

3. Shuttle Have two origins of YEp (Yeast Episomal For gene cloning
Vectors replication and selectable Plasmid), pDual, Ti-based and transfer
markers for replication in binary vectors between two
two different hosts (e.g., different species.
bacteria and yeast).

4. Yeast Designed for use in YEp, YRp, YCp, YIp, YAC For cloning,
Vectors Saccharomyces cerevisiae; expression, and
may replicate episomally or genetic studies in
integrate into yeast genome. yeast.

5. Viral Modified viruses that carry Adenoviral, Retroviral, For gene delivery
Vectors foreign genes into host cells; Lentiviral, Baculovirus and therapeutic
 used in gene therapy and vectors applications.
transfection.

6. Artificial Large-capacity vectors that BAC (Bacterial Artificial For mapping and
Chromosome can carry very large DNA Chromosome), YAC (Yeast cloning large
s inserts. Artificial Chromosome), genomes.
MAC (Mammalian Artificial
Chromosome)

7. Cosmids Hybrid between plasmid and λ Cosmid pJB8, pHC79 For cloning
phage; contain cos sites for medium-to-large
packaging into phage DNA fragments
particles. (~45 kb).

8. Phagemid Combine features of plasmids pBluescript, pUC119 Useful in

Vectors and filamentous phages (like sequencing and
M13); can produce single- mutagenesis.
stranded DNA.
NATURAL VECTORS
Category Examples Description / Use

Plasmids F plasmid, R Naturally occurring circular DNA molecules in

plasmid bacteria that replicate independently of the
chromosome.

Bacteriophages λ (lambda) phage, Viruses that infect bacteria and can transfer their
(phage vectors) M13 phage DNA into the host cell.

Ti plasmid From Agrobacterium Transfers T-DNA into plant genomes — naturally

tumefaciens used for plant genetic transformation.

Transposons Tn elements Mobile DNA segments that can move within or

between DNA molecules.

ARTIFICIAL VECTORS
Category Examples Description / Use

Plasmid cloning pBR322, pUC18/pUC19, pBluescript Engineered plasmids with ori,

vectors MCS, and selectable markers for
easy cloning.

Yeast vectors YEP (Yeast Episomal Plasmid), YRP Designed for gene cloning and
(Yeast Replicating Plasmid), YAC expression in yeast.
(Yeast Artificial Chromosome)

Bacterial Artificial pBAC, F-factor-based vectors Used for cloning large DNA
Chromosomes fragments (~300 kb) in
(BACs) bacteria.

Expression vectors pET series, pGEX, pMAL Modified to express foreign

proteins under specific
promoters.

Viral vectors Retroviral, Adenoviral, Lentiviral Engineered viruses used in

vectors gene therapy to deliver genes
into animal or human cells.

Cosmids (partly Derived from λ phage Combine plasmid and phage

natural) features — allow larger DNA
inserts.
Examples of Vectors
pBR322
 pBR322 is a commonly used plasmid cloning vector used in prokaryotes, primarily E.
coli.
 The vector consists of an origin of replication from a ColE1-like plasmid, pMB1, an ApR
gene (Ampicillin resistance gene) from the transposon, Tn3, and a TcR gene from
pSC101.
 pBR322 was designed to overcome the limitations with pBR312 and pBR313, both of
which have extraneous DNA sequences and restriction enzyme cleavage sites that
affected their function as vectors.
 The structure of pBR322 was designed to maximize the number of restriction enzyme
cleavage sites on the vector and to minimize its size.
 The vector contains twenty-one unique restriction enzyme cleavage sites, eleven of
which are present in the TcR and ApR genes.
 The structure also facilitates a unique EcoRI cleavage site within the plasmid in order
to increase the efficiency of the vector.
 The pBR322 family of vectors was initially created for general cloning purposes in E.
coli and other similar prokaryotes; however, over the years, derivatives of the vector
have been designed for cloning purposes that specific to a particular organism or a
particular function.
 Even though pBR322 has been used for decades as an effective multipurpose cloning
vector, it has some limitations.
 The vector might be lost in continuous culture in the absence of selective pressure,
which might be a problem in large-scale fermentation of recombinant bacteria.

Feature pBR322 pBR325 pBR326 pBR327

Definition A small, artificial A derivative of A pBR322- A smaller
cloning vector pBR322 containing derived vector derivative of
developed by an extra carrying ampᴿ pBR322
Bolivar & chloramphenicol and chlᴿ, used containing
Rodriguez (1977), resistance (chlᴿ) for dual tetᴿ and chlᴿ
used in E. coli for gene for triple- selection (no genes; lacks
recombinant DNA antibiotic selection. tetracycline). ampicillin
experiments. resistance.
Size (kb) ~4361 ~5995 (~6kb) ~4.8 ~4.2
Antibiotic ampᴿ, tetᴿ ampᴿ, tetᴿ, chlᴿ ampᴿ, chlᴿ tetᴿ, chlᴿ
Resistanc
e Genes
Copy Moderate (~15– Moderate (~15–20 Moderate Moderate
Number 20 per cell) per cell)
Cloning / Within ampᴿ (PstI) Within ampᴿ, tetᴿ, Within ampᴿ Within tetᴿ
Restriction and tetᴿ (BamHI, and chlᴿ and chlᴿ and chlᴿ
Sites HindIII, SalI, etc.)
Relative Smallest of all Largest Medium Small
Size
Key Base vector for Multi-antibiotic Dual resistance; Compact
Features / many derivatives; selection and co- simple for vector; useful
Uses classic cloning transformation cloning when
vector studies ampicillin not
 required

All four plasmids share the pMB1 origin of replication and allow easy detection of
recombinants via insertional inactivation of resistance genes. Low copy number limits
large-scale DNA production, so modern high-copy vectors like the pUC series are
preferred for high-yield cloning.

pUC Series: pUC18, pUC19, pUC118, pUC119

 pUC19 is also an example of a plasmid cloning vector that is
used for the transfer of recombinant DNA fragments into a
host cell.
 The name ‘pUC19’ is given to the vector where the ‘p’
indicates plasmid and ‘UC’ indicates the University of
California’ where the vector was designed and constructed.
 The vector has been extensively used for cloning purposes
where the host cells containing the plasmid are distinguished from the ones that do
not have it by the differences in the color of the
colonies on the growth medium.
 The vector is a double-stranded DNA molecule with
2686 bp and a high copy number.
 pUC19 consists of a 54 base-pair cloning site polylinker
that further contains 13 different hexanucleotide-
specific restriction endonucleases.
 Selectable Marker is ampᴿ (ampicillin resistance) for all.
 Multiple Cloning Site (MCS) Orientation: pUC18 and
pUC19 contain the same set of restriction sites within
the MCS, but the orientation of this site is reversed. The
MCS of pUC118 is inserted in the same orientation as the M13mp18 phage, allowing
single-stranded DNA production when needed.
(pUC119, its counterpart, carries the MCS in the opposite orientation.)
 Functionality: This difference is useful for cloning DNA inserts in either direction. By
choosing either pUC18 or pUC19, researchers can control the orientation of the
inserted fragment.
 Screening: lacZ α-complementation:
 o Functional α-fragment + ω-fragment → β-galactosidase → cleaves X-gal → blue
colonies
o Insert disrupts α-fragment → white colonies
IPTG induces lac promoter; X-gal provides colorimetric readout. Same for all.
Other features: Otherwise, the plasmids are identical in other key aspects, such as their
small size, high copy number, and use of ampicillin resistance as a selectable marker

Feature pUC18 pUC19 pUC118 pUC119

Definition A small, high- Identical to A derivative of A derivative of
copy-number pUC18 but with pUC18 pUC19 containing
cloning vector the multiple containing an an M13 origin
derived from cloning site M13 origin of of replication,
pBR322 and (MCS) replication for allowing
M13mp19, sequence single-stranded preparation of
designed for easy reversed in DNA both double- and
blue-white orientation. production. single-stranded
screening in E. DNA.
coli.
Size (kb) 2686 kb 2686 kb ~3.2 kb ~3.2 kb
Origin of pMB1 ori pMB1 ori pMB1 ori + pMB1 ori + M13
Replication (mutated to yield (same) M13 ori ori
(ori) high copy
number)
Copy High (>500 High (>500 High (similar to High (similar to
Number copies per cell) copies per cell) pUC18) pUC19)
Multiple Located within Same sites as Same as pUC18 Same as pUC19
Cloning lacZ’ gene (in pUC18 but + M13 origin + M13 origin,
Site (MCS) the same reversed
orientation as orientation
M13mp18)
Single- No No Yes (contains Yes (contains M13
Stranded M13 ori) ori)
DNA
Production
Key Standard pUC MCS reversed pUC18 + M13 pUC19 + M13 ori
Differences vector compared to ori → single- → single-stranded
from Each pUC18 stranded DNA DNA possible
Other possible
Application Routine cloning, Same as Used for Used for cloning
s sequencing, pUC18, cloning and and ssDNA
subcloning compatible with ssDNA sequencing
opposite MCS sequencing
orientation
T/A Cloning Vector
1. pDrive Vector
The pDrive vector is a T/A cloning vector supplied by
Qiagen for rapid cloning of PCR products.
It is designed for direct insertion of DNA fragments
amplified by Taq polymerase (with A-overhangs).
Key Features:
 T-overhangs at 3′ ends for easy ligation of A-tailed
PCR products.
 Ampicillin and Kanamycin resistance genes for
selection.
 lacZ α-fragment for blue-white screening of recombinants.
 High-copy-number plasmid with pUC origin of replication for easy plasmid yield.
pGEM-T Easy Vector
The pGEM-T Easy vector (Promega) is another T/A cloning vector used for cloning PCR
products.
It contains single 3′-T overhangs at the insertion site for direct ligation of A-tailed DNA
fragments.
Key Features:
 T-overhangs for T/A cloning.
 Ampicillin resistance gene (Amp^R) for
selection.
 lacZ α-peptide region for blue-white screening.
 Multiple Cloning Site (MCS) within the lacZ
region for easy insertion analysis.
 High-copy-number pUC origin of replication for
efficient plasmid amplification.

Applications of Vectors
The application of vectors in molecular biology and
genetic engineering has increased with time due to
the simplicity, cost-effectiveness, and rapidity of
the process. The following are some of the major applications of vectors in molecular
biology;
1. Cloning vectors are the most important group of vectors that are used for the
transfer of foreign DNA into host cells for different purposes.
2. One of the most important applications of vectors is to generate engineered
organisms for a particular function, like engineering E. coli bacteria for insulin
production.
3. Vectors can be used to isolate a particular gene sequence within a genome and to
determine its nucleotide sequence through DNA sequencing.
4. It also helps determine control sequences and regulatory sequences in genomes for
their study and analysis.
5. Cloning vectors can be used for studying the structure, function, and production of
protein in different organisms.
 6. Phage therapy is a form of therapy that uses bacteriophage vectors to treat
different bacterial infections in humans and other animals.
7. Vectors can also be used to identify mutations in different regions of DNA
sequences as well as to diagnose gene defects related to certain diseases.
8. Recombinant DNA technology has been used in clinical microbiology in different
approaches like recombinant antigens, recombinant vaccines, and diagnostic
probes.
9. Recombinant antigens prepared by cloning techniques by using cloning vectors
have been used for the screening of diseases like HIV, HCV, and CMV.
10. Vectors are one of the components in molecular biology which enable
numerous studies related to cell structure, nucleic acid composition, and genetic
engineering techniques.

Limitations of Vectors
The following are some of the limitations of vectors;
1. Vectors are not very stable due to changes in metabolic energy and changing pH
and temperature in different hosts. The stability of vectors depends largely on the
type of vector and host genotypes.
2. Overexpression of a particular type of genes in the host cell is a common problem
associated with the use of vectors.
3. The use of a single type of vector might not be sufficient for a particular purpose.
The use of multiple vectors is complex and results in difficulties during the process.
4. Even though a large number of studies are done in the field of molecular biology for
the production of more efficient vectors, it is a time-consuming and expensive
process.
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PLASMID
A plasmid is a small, circular, double-stranded DNA molecule found in bacteria and some
archaea and eukaryotes, that replicates independently of the chromosomal DNA. Plasmids
are typically non-essential for basic survival but can carry beneficial genes that provide
selective advantages under specific conditions.
Plasmids are widely used in molecular biology and genetic engineering due to their ability
to carry and transfer genetic material.

A typical plasmid DNA is considered to be less than 5% the size of the bacterial
chromosome being 1kb-250kb+. Most of the plasmid DNA isolated from bacterial cells
exist in the supercoil configuration, which is the most compact form for DNA to exist
within the cell. The copy number refers to the fact that different plasmids occur in cells in
different numbers. Some plasmids are present in the cell in only 1-3 copies, whereas
others may be present in over 100 copies. Copy number is controlled by genes on the
plasmid and by interactions between the host and the plasmid.

Plasmids encode only few proteins required for their own replication (replication proteins)
and these proteins encoding genes are located very close to the ori. All the other proteins
required for replication, e.g. DNA polymerases, DNA ligase, helicase, etc., are provided by
the host cell. Thus, only a small region surrounding the ori site is required for replication.
Other parts of the plasmid can be deleted and foreign sequences can be added to the
plasmid without compromising replication.

HISTORICAL BACKGROUND
 1946: Joshua Lederberg and Edward Tatum demonstrated bacterial conjugation, where
genetic material is transferred between bacteria.
 1952: The term "plasmid" was introduced by Lederberg to describe extrachromosomal
genetic elements.
 1960s: Discovery of R (resistance) plasmids that carry genes for antibiotic resistance.
 1973: Boyer and Cohen utilized plasmids in recombinant DNA experiments, leading to
a revolution in biotechnology and gene cloning.

CLASSIFICATION OF PLASMIDS
Based on Function:
1. Fertility Plasmids (F plasmids or Episomes)
Fertility plasmids contain tra (transfer) genes, including those for the formation of sex pili,
enabling bacterial conjugation—a mechanism of horizontal gene transfer. F-plasmid is a
circular dsDNA molecule of ~1kb.
The F factor is a well-known fertility plasmid in Escherichia coli. It can exist in multiple
states:
 F⁺ (F-positive): Contains the F plasmid independently in the cytoplasm. These cells can
form a conjugation pilus and donate the F factor to F⁻ cells.
 F⁻ (F-negative): Do not have the F plasmid. These act as recipient cells during
conjugation.
 Hfr (High Frequency of Recombination): The F plasmid is integrated into the bacterial
chromosome. During conjugation, these cells attempt to transfer the F factor along
with adjacent chromosomal DNA, often leading to recombination in the recipient cell.
Why “High Frequency”?
Because chromosomal genes near the site of F integration are transferred to recipients
at a much higher frequency than in F⁺ to F⁻ conjugation, making Hfr cells valuable
tools for chromosomal gene mapping.
 F′ (F-prime): Result from improper excision of the F factor from the chromosome in Hfr
cells, bringing with it adjacent bacterial genes. These F′ plasmids can be transferred
during conjugation, allowing specialized transduction of bacterial genes.

2. Resistance (R) Plasmids

R plasmids carry and transmit genes for antibiotic resistance from one cell to another.
These resistance genes protect bacteria from antibiotics used in human and veterinary
medicine as well as antibiotics naturally present in soil microorganisms. In addition to
antibiotics, some R plasmids also provide resistance to heavy metals (e.g., mercury,
arsenic, cadmium) or other toxic compounds.
Usually large (often 20 kb to over 200 kb). Typically, low-copy plasmids (1–5 copies per
cell). Contains bla gene: Encodes β-lactamase, conferring resistance to penicillin. tetA:
Encodes tetracycline resistance.

Example: R100: A well-known plasmid carrying multiple resistance genes.

R plasmids often have conjugation systems, allowing rapid spread of resistance among
bacterial populations, even across species.

3. Bacteriocinogenic/ Colicin (Col) Plasmids

These plasmids encode bacteriocins, which are protein toxins that kill or inhibit other
closely related bacteria, providing a competitive advantage to the host. Usually small to
medium (5–30 kb).
Often moderate to high copy plasmids (10–50 copies per cell).
Example: ColE1 plasmid in E. coli encodes colicin E1, which disrupts the membrane
potential of susceptible bacteria. Features:
 Also encode immunity proteins that protect the host cell.
 Can be conjugative or non-conjugative.
 Provide a competitive advantage by killing off rival strains, helping the host bacterium
dominate in a mixed microbial community.
4. Metabolic/Degradative Plasmids
These plasmids allow bacteria to metabolize unusual or complex organic compounds that
are not normally used as carbon or energy sources. Examples include the ability to digest
toluene, camphor, salicylic acid, naphthalene, and petroleum hydrocarbons (“oil-eating
bacteria”).
Often large (80–200 kb). Usually low copy plasmids.
Common in environmental and soil bacteria like Pseudomonas spp., these plasmids
enable survival in polluted or nutrient-limited conditions.

5. Virulence Plasmids
Virulence plasmids carry virulence factors—genes that enhance a microbe’s ability to
cause disease. These factors may include toxins, adhesion proteins, secretion systems, or
other traits that help pathogens colonize, evade host defenses, and damage host tissues.
virulence factor is any molecule or trait produced by a microorganism (bacterium, virus,
fungus, or parasite) that helps it infect a host, evade defenses, or cause disease.
Variable in size, often 30–200 kb. Usually low copy plasmids.
Examples:
 Ti plasmid in Agrobacterium tumefaciens causes crown gall disease in plants via
transfer of T-DNA into plant cells.

 Virulence plasmids in Shigella → carry genes for invasion proteins that allow entry into
epithelial cells.
These plasmids play crucial roles in bacterial diseases of humans, animals, and plants.

6. Cryptic Plasmids
Cryptic plasmids are plasmids with no known or observable phenotype. They may still
provide advantages under specific or stress conditions or serve as evolutionary reservoirs
of genetic material.
They are often small and do not carry genes for transfer or antibiotic resistance but are
stably inherited.

Based On Mobility
Type Description
Conjugative Contain tra genes and can transfer themselves via conjugation.
Plasmids
Non-conjugative Lack tra genes; cannot transfer themselves, but may be
Plasmids mobilized by conjugative plasmids.
PLASMID STRUCTURE
Plasmids are small, circular, double-stranded DNA molecules that exist separately from a
cell's chromosomal DNA. While primarily found in bacteria, they can also be present in
archaea and eukaryotes. Plasmids replicate autonomously from the chromosome and
often carry genes that confer advantages to the host cell, such as antibiotic resistance or
metabolizing uncommon substances.

1. Every plasmid has certain essential elements. These are as follows –

 Origin of replication (OR) – This refers to a specific location in the strand where the
replication process begins. In plasmids, this region is A=T rich region as it is easier to
separate the strands during replication.
 Selectable marker site – This region consists of Antibiotic resistance genes which
are useful in the identification and selection of bacteria that contain plasmids.
 Promoter region – this is the region where the transcriptional machinery is loaded.
 Primer binding site – this is the short sequence of single-strand DNA which is useful
in DNA amplification and DNA sequencing.
 Multiple cloning sites – This site contains various sequences where the restriction
enzymes can bind and cleave the double stranded structure.
 Addiction Systems (toxin-antitoxin): Kill cells that lose the plasmid, ensuring
maintenance.
2. The size of the plasmid varies from 2 kb to 200 kb.
3. It is the extrachromosomal element of the cell, not required for the growth and
development of the cell.
4. Most of the plasmids contain the TRA gene, which is the transferred gene and is
essential in transferring the plasmid from one cell to another. Partitioning Systems (par
genes): Ensure equal plasmid distribution during cell division.

PLASMID ISOLATION FROM BACTERIAL CELLS

Step 1: Cell Lysis
 Purpose: To break open bacterial cells and release plasmid and chromosomal DNA into
solution.
 Process: The bacterial cells are treated with lysozyme (which digests the peptidoglycan
layer of the cell wall) and SDS (sodium dodecyl sulfate), a detergent that dissolves the
cell membrane and denatures proteins.
 Result: Cellular contents, including plasmid DNA, chromosomal DNA, and proteins, are
released.
Step 2: Centrifugation
 The lysate is centrifuged to remove large cell debris and insoluble materials.
 Result: The supernatant contains plasmid DNA, chromosomal DNA, RNA, and soluble
proteins.

Step 3: Plasmid DNA in Supernatant

 After centrifugation, the plasmid DNA remains in the supernatant, as it is small and
soluble compared to larger cell fragments that pellet down.

Step 4: Treatment with Ethidium Bromide and Cesium Chloride (CsCl₂)

 Principle: A density gradient is formed using CsCl and ethidium bromide (EtBr).
 Function:
o Ethidium bromide intercalates (inserts) between the base pairs of DNA.
o It binds more with linear chromosomal DNA than with supercoiled plasmid DNA.
o This difference in binding changes the buoyant density of each DNA type.
 Result: Plasmid and chromosomal DNA acquire different densities, allowing separation
during ultracentrifugation.

Step 5: Denaturation (Density Adjustment)

 The DNA mixture is gently heated or chemically treated to partially denature linear
DNA, further increasing its density difference from the supercoiled plasmid DNA.

Step 6: Ultracentrifugation
 The tube is centrifuged at high speed (in an ultracentrifuge) for several hours.
 A density gradient forms due to CsCl₂, and DNA molecules migrate to the point where
their density matches that of the surrounding solution.

Step 7: Formation of DNA Bands

 After centrifugation, distinct bands appear in the gradient:
o Upper band: Supercoiled plasmid DNA (lower density).
o Lower band: Linear or nicked chromosomal DNA (higher density).
 Ethidium bromide makes these bands visible under UV light.

Step 8: Isolation of Plasmid DNA

 The uppermost band, containing plasmid DNA, is carefully removed using a syringe or
pipette.
 Ethidium bromide is then removed by extraction with isoamyl alcohol or similar
solvents, and the plasmid DNA is precipitated with alcohol (ethanol or isopropanol).
 Finally, the purified plasmid is dissolved in a suitable buffer (e.g., TE buffer).

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