Vectors in Biology: Types, Roles, and Significance in Gene Cloning

Vectors are essential tools in gene cloning, serving as carriers to transfer foreign DNA into host cells for replication and expression. They enable genetic manipulation in biotechnology, medicine, and research. Different types of vectors include plasmids, small circular DNA molecules that replicate independently in bacteria; bacteriophages, viruses like lambda phage used for high-efficiency DNA transfer; and cosmids, hybrid vectors combining plasmid and phage properties to carry larger DNA fragments. Additionally, artificial chromosomes such as Yeast Artificial Chromosomes (YACs) and Bacterial Artificial Chromosomes (BACs) allow cloning of large DNA sequences, crucial in genome studies. Viral vectors, including retroviruses and adenoviruses, are widely used in gene therapy and molecular biology. Each vector type has unique advantages and applications, making them fundamental tools in recombinant DNA technology and genetic engineering.

Vectors are crucial tools in gene cloning, acting as vehicles that carry and introduce foreign DNA into host cells for replication, expression, and further genetic manipulation. They play a fundamental role in recombinant DNA technology, enabling advancements in biotechnology, medicine, and genetic research. Various types of vectors serve different purposes based on their structure and capacity. Plasmids are small, circular DNA molecules that replicate independently in bacterial cells, making them widely used for gene cloning due to their simplicity and efficiency. Bacteriophages, such as lambda phage, are viruses that infect bacteria and offer high-efficiency gene transfer, making them suitable for cloning larger DNA fragments. Cosmids are hybrid vectors that combine plasmid and bacteriophage properties, allowing them to carry even larger DNA sequences while maintaining high cloning efficiency. Artificial chromosomes, including Yeast Artificial Chromosomes (YACs) and Bacterial Artificial Chromosomes (BACs), are used to clone very large DNA fragments, making them essential tools for genome mapping and sequencing projects. Viral vectors, such as retroviruses and adenoviruses, are engineered to deliver genes into eukaryotic cells and are widely applied in gene therapy and molecular biology research. Each vector type has distinct advantages, limitations, and applications, making them indispensable in genetic engineering, biotechnology, and medical advancements, including the development of new treatments for genetic disorders, production of recombinant proteins, and synthetic biology innovations.

1. Vectors and Their
Types in Gene Cloning

By Muhammad Umar

A department of Biotechnology

2. VECTOR AND ITS TYPES FOR GENE
CLONING
___
Keywords: gene cloning, vectors, plasmids, cosmids, artificial chromosomes

In molecular cloning, a vector is like a tool or carrier (such as plasmids, cosmids, or Lambda phages) used to
transfer a piece of foreign genetic material, usually DNA, into another cell.
Inside the new cell, this DNA can be copied or used to make proteins. When a vector carries foreign DNA, it is called
recombinant DNA. There are four main types of vectors: plasmids, viral vectors, cosmids, and artificial chromosomes.
Among these, plasmids are the most commonly used. All vectors are specially designed and include three key features:
a starting point for copying DNA (called the origin of replication), a site where the foreign DNA can be added (called the
multicloning site), and a marker to identify the cells that have successfully received the vector.
A vector usually has two parts: the insert (which is the DNA we want to transfer) and the backbone (the main structure
of the vector). The purpose of a vector is to transfer the genetic material into another cell, where it can either be copied
many times or used to produce proteins. All vectors can be used for cloning, but some are designed specifically for
certain tasks. For example, expression vectors are made to produce proteins in the target cell. They contain a promoter,
a sequence that helps the DNA to be read and used to make proteins. On the other hand, transcription vectors are only
meant to copy the DNA; they cannot produce proteins. These vectors are often used when scientists need many copies
of the DNA but do not need it to make proteins.
Scientists usually work with vectors in E. coli (a type of bacteria) because these vectors are designed to survive and
work well in these cells. However, some vectors can also work in other organisms like yeast, plants, or animal cells.
These are called shuttle vectors because they can move between different types of organisms. Shuttle vectors may
include elements from bacteria or viruses to help them work in the new host. Some newer vectors, called intragenic
vectors, are designed to avoid using genetic material from unrelated species. When a vector is added to a target cell,
the process is called different names depending on the type of cell. For bacterial cells, it is called transformation. For

3. eukaryotic cells (like plant or animal cells), it is called transfection. If a virus is used to carry the vector, the process is
called transduction.
Three features of all cloning vectors
1. Small in size.
2. Sequences that permit the propagation of itself in bacteria or in yeast (The replication origin).
3. A cloning site to insert foreign DNA; the most versatile vectors contain a site that can be cut bymany restriction
enzymes.
Multiple Cloning Site (MCS):
A method of selecting for bacteria or yeast containing a vector with foreign DNA; usually accomplished by selectable
markers for drug resistance or/and Reporter genes.

a b
Figure 1: (a)diagram of a cloning vector,(b) common cloning vector pbr332 in E.coli
The replication origin (ORI):
The replication origin (ORI) is a specific DNA sequence of 50 – 100 base pairs that must be present in a plasmid
for it to replicate.
Host-cell enzymes bind to ORI, initiating replication of the circular plasmid.
Once DNA replication is initiated at ORI, it continues around the circular plasmid regardless of its nucleotide
sequence.
Thus any DNA sequence inserted into such a plasmid is replicated along with the rest of the plasmid DNA.
Selectable marker:
A selectable marker is carried by the vector to allow the selection of positively transformed cells. Antibiotic resistance
is often used as marker, an example is the beta-lactamase gene which confers resistance to the penicillin group of
beta-lactam antibiotics like ampicillin.
Scientists usually work with vectors in E. coli (a type of bacteria) because these vectors are designed to survive and
work well in these cells. However, some vectors can also work in other organisms like yeast, plants, or animal cells.
These are called shuttle vectors because they can move between different types of organisms. Shuttle vectors may
include elements from bacteria or viruses to help them work in the new host. Some newer vectors, called intragenic
vectors, are designed to avoid using genetic material from unrelated species. When a vector is added to a target cell,
the process is called different names depending on the type of cell. For bacterial cells, it is called transformation. For
eukaryotic cells (like plant or animal cells), it is called transfection. If a virus is used to carry the vector, the process is
called transduction.

4. TYPES OF CLONING VECTORS
1. Plasmids:
plasmids are small, extra chromosomal DNA molecules that are stably inherited from one generation to another in the
extra chromosomal DNA state. usually closed circles of either single stranded or double stranded DNA. plasmids are
widely distributed throughout prokaryotes and range in size from approximately 15 hundred dp to 300 kbp. plasmids
are commonly dispensable i.e. not essential to their host cells and mot all cells contain plasmids. for example,
plasmids are commonly absent in bacteria of the genre, Brucella Rickketsia.
the replication of the plasmids is often coupled to that of the host cell in which it is maintained, with the plasmid
replication occurring at the same time as the host genome is replicated. they contain an origin of replication, which
enables them to be replicated independently, although this normally realize on polymerases and other component of
host cell's machinery.
Conjugative Plasmids: Plasmids contain, tra genes, genus (genes necessary for non-sexual transfer of genetic material)
which perform the complex process of conjugation the transfer of plasmids to other bacteria e.g. all f and f' plasmids,
may R plasmids, and some col plasmids are conjugative plasmids.
Non-Conjugative Plasmids: These plasmids are incapable of initiating conjugation hence they can only be transferred
with the assistance of conjugative plasmids.
On the other hand, plasmids used for making proteins are called expression vectors. These plasmids have extra
features, such as a ribosome binding site and start and stop codons, which allow the cell to translate the DNA into a
protein.
Plasmid Map: A plasmid map is a graphical representation of
a circular DNA molecule
commonly used in genetic engineering and molecular
biology. It illustrates the
plasmid's structural features, such as the origin of
replication (Ori), selectable
markers (e.g., antibiotic resistance genes),
restriction enzyme sites, promoters, coding sequences, and
other functional elements.
These maps are essential for understanding the
plasmid's design, facilitating cloning, expression, or
other molecular biology experiments.
Figure 1: Map of a Plasmid (cloning vector)
2. Viral Vectors:
Viral vectors work like a “nano syringe” to deliver nucleic acids to a target. They are often more efficient
than other transfection methods, are useful for whole organism studies, have a relatively low toxicity, and
are promising candidates for human gene transfer.
All viral vectors require a host for replication. The production of a viral vector is typically separated from the
ability of the viral vector to cause disease. While viral vectors maintain their ability to “infect” cells, most
viral vectors are replication deficient under experimental conditions (although there are some replication
competent viral vectors in use). Therefore, replication deficient viral vectors are not capable of establishing
a productive disease and are generally not considered to be infectious biological agents.

5. It is important to note that if a viral vector can transduce a human cell line in the lab, it can likely deliver
genetic material to YOUR cells upon accidental exposure. Viral vectors should always be handled with
caution.
Viral techniques use various classes of viruses as a tool for gene delivery. Viruses introduce their DNA into
the cells with high efficiency. Therefore it is possible to take advantage of this by introducing a foreign gene
into the virus and then using the properties of the virus to deliver this gene with high efficiency into the
target cells.
Gene therapy vectors are being developed by genetic modification of retroviruses, adenoviruses, poxviruses,
parvoviruses (adeno-associated viruses), herpesviruses, and others. Unlike wild type viruses, these vectors
are used to transfer therapeutic genes into target cells and thus are engineered by deleting the essential
genes which allow replication, assembling, or infection. These genes can be replaced by a therapeutic gene
to make the genome of a gene therapy vector. Such vectors lose their ability to reproduce in target cells and
can be replicated only in a cell line which supplies the deleted function. Replication deficiency ensures the
safety of viral vectors, but on the other hand, vectors need to be produced in large amounts of virus
particles. For this purpose, there are specialized cell lines called “packaging cell lines” (PCLs) engineered to
replace a function of a deleted viral gene and for the production of recombinant viruses.
ADENOVIRUS GENERAL DESCRIPTION:
Adenoviruses are non-enveloped, icosahedral, viruses with a linear, double-stranded DNA genome of
approximately 36kb with a lytic infection cycle. There are 57 immunologically distinct types of adenovirus
that can cause human infection. Recombinant adenoviruses used for biomedical research are primarily
based on Adenovirus 5.
HERPES VIRUS GENERAL DESCRIPTION:
Herpes Simplex Virus (Types I and II) are icosahedral, lipid enveloped, double-stranded linear DNA viruses
approximately 110- 200nm in diameter. HSV types I and II can be differentiated immunologically. HSV-I is
herpes gingivostomatitis; whereas HSV-II is herpes genitalis, or genital herpes. HSV-derived vectors are
unique in that the vectors have a wide host range and cell tropism in dividing and non-dividing cells, and are
able to infect almost every cell type in most vertebrates.
3. COSMIDS:
A cosmid is a plasmid that contain phage sequence that allows the vector to be packaged and transmitted to
bacteria like phage vector.
A cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid with a bacterial "ori"
sequence and a "cos" sequences derived from the lambda phage. Cos site is the sequence required by
a DNA molecule in order to be recognized as a "2 genome' by the proteins that package DNA into 2
phage particles.
Cosmid DNA containing particles are as transmittable as real λ phages, but once inside the cell, the
cosmid cannot control synthesis of new phage particles and instead replicates as a plasmid.
Recombinant DNA is therefore obtained from colonies rather than plaques. They frequently also contain
a gene for selection such as antibiotic resistance.
They are able to load 37 to 52 kb of DNA, while normal plasmids are able to carry only 126 kb.
Sometimes helper phage is used to assist in packaging of cosmid inside phage. Helper phage provides
the essential proteins required for packaging which are lacked by cosmid vector.

6. For packaging into a phage, concatemer formation is required (cosmid-insert- cosmid).
This is generated by using two cos sites flanked by the insertion site for foreign DNA. Providing the
inserted DNA in the right size, in vitro packaging cleaves the cos sites and replaces the recombinant
cosmids in mature phage particles.

Figure 1. Outline of procedures used in cloning with a cosmid vector. This vector contains a cos site, a restriction site for inserting exogenous DNA, and a gene for ampicillin resistance.
Exogenous DNA is cut with an appropriate restriction enzyme, as is the vector. The vector and exogenous DNA are ligated together, producing a recombinant molecule of 37–52 kb that
can be packaged in λ by in vitro packaging. The packaged vector infects E. coli, injecting its DNA into the host, where it circularizes and multiplies. Escherichia coli cells that receive the
cosmid are distinguished from cells that are not infected by their ability to survive on media containing ampicillin.
4. Artificial Chromosomes:
• Artificial chromosomes are DNA molecules assembled in vitro from defined constituents,which
guarantee stable maintenance of large DNA fragments with the properties of natural chromosomes.
• Artificial chromosomes are useful for genome sequencing programmes, for functional characterization
of entire genomic regions and for the transduction of large DNA segments into human and nonhuman
mammalian cells. Types: BAC, YAC, PAC, HAC
Bacterial artificial chromosome:
A bacterial artificial chromosome (BAC) is an engineered DNA molecule used to clone DNA sequences in
bacterial cells (for example, E. coli). BACs are often used in connection with DNA sequencing. Segments of an
organism's DNA, ranging from 100,000 to about 300,000 base pairs, can be inserted into BACs. The BACs,
with their inserted DNA, are then taken up by bacterial cells. As the bacterial cells grow and divide, they
amplify the BAC DNA, which can then be isolated and used in sequencing DNA.
A large piece of DNA can be engineered in a fashion that allows it be propagated as a circular artificial
chromosome in bacteria--so-called bacterial artificial chromosome, or BAC. Each BAC is a DNA clone
containing roughly 100 to 300 thousand base pairs of cloned DNA. Because the BAC is much smaller than the
endogenous bacterial chromosome, it is straightforward to purify the BAC DNA away from the rest of the
bacteria cell's DNA, and thus have the cloned DNA in a purified form. This and other powerful features of
BACs have made them extremely useful for mapping and sequencing
mammalian genomes.
Applications of BAC:

Contribution to models of disease: Inherited disease
• BACS are now being utilized
in modeling geneticdiseases, often
alongside transgenic mice.
• BACS have been useful in
this field as complexgenes may have

7. several regulatory sequences upstream of the encoding
sequence, including various promoter sequences that
will govern a gene's expression level.
BACS have been used to study neurological diseases
such as Alzheimer's disease or as in the case of
aneuploidy associated with Down syndrome. There
have also been instances when they have been used to
study specific oncogenes associated with cancers.

Figure 1: This diagram illustrates the process of inserting a
gene of interest into a bacterial plasmid and transferring it
into Escherichia coli bacteria, a
common method in molecular biology for cloning or
expressing genes.
Applications of BAC [contd.]
i. Contribution to models of disease: Infectious disease
The genomes of several large DNA viruses and RNA viruses have been cloned as BACS.
These constructs are referred to as "infectious clones".
The infectious property of these BACS has made the study of many viruses such as the herpesviruses, poxviruses
and coronaviruses more accessible.
Yeast artificial chromosome:
It is a human-engineered DNA molecule used to clone DNA sequences in yeast cells. Segments of an
organism's DNA, up to one million base pairs in length, can be inserted into YACS.
Yeast artificial chromosomes (YACs) are plasmid shuttle vectors capable of replicating and being selected in
common bacterial hosts such as Escherichia coli, as well as in the budding yeast Saccharomyces cerevisiae.
They are of relatively small size (approximately 12 kb) and of circular form when they are amplified or
manipulated in E. coli, but are rendered linear and of very large size, i.e. several hundreds of kilobases (kb),
when introduced as cloning vectors in yeast.
The YACS, with their inserted DNA, are then taken up by yeast cells. As the yeast cells grow and divide, they
amplify the YAC DNA, which can be isolated and used for DNA mapping and sequencing.
The latter process involves cleavage at strategically located sites by two restriction enzymes, which break
them in two linear DNA arms. These are subsequently ligated with the appropriate DNA insert before
transformation into recipient yeast cells. In this linear form, these specialized vectors contain all three cis-
acting structural elements essential for behaving like natural yeast chromosomes: an autonomously
replicating sequence (ARS) necessary for replication; a centromere (CEN) for segregation at cell division; and
two telomeres (TEL) for maintenance.

8. Expression of a Vector:
Transcription:
It is the synthesis of an mRNA molecule from a DNA template All the cellular RNAs are synthesized from the DNA
templates through the process the transcription, DNA regions that can be transcribed into RNA are called structural
genes Template strand on which the RNA is synthesized is called as antisense code or non-sense codon, the coding
strand is known as sense codon, the enzyme that catalyze the RNA synthesis is RNA Polymerase and doesn't need a
primer unlike replication.
An expression vector must have elements necessary for protein expression. These may include a
strong promoter, the correct translation initiation sequence such as a ribosomal binding site and
start codon, a strong termination codon, and a transcription termination sequence. Where the
promoter is present, the expression of the gene is preferably tightly controlled and inducible so that
proteins are only produced when required. Protein expression may also be also constitutive(i.e.
protein is constantly expressed) in some expression vectors. Low level of constitutive protein
synthesis may occur even in expression vectors with tightly controlled promoters.
constitutive protein synthesis may occur even in expression vectors with tightly controlled promoters.
What are the risks of cloning?
• Reproductive cloning is expensive and highly inefficient
• Cloned animals tend to have more compromised immune function and higher rates of infection, tumor growth, and
other disorders
• Genomes of cloned mice are compromised, 4% of genes function abnormally
• The abnormalities do not arise from mutations in the genes but from changes in the normal activation or
expression of certain genes.
• A process called "imprinting" chemically marks the DNA from the mother and father so that only one copy of a
gene (either the maternal or paternal gene) is turned on. Defects in the genetic imprint of DNA from a single donor
cell may lead to some of the developmental abnormalities of cloned embryos
Conclusion:
In conclusion, various types of vectors play crucial roles in genetic research and biotechnology. Plasmids, viral vectors,
and artificial chromosomes are all tools used to carry and express foreign genes in host cells. Plasmids are often used
for cloning, while viral vectors can be used for both gene transfer and protein production. Artificial chromosomes,
such as YACS, BACs, and HACs, allow for the integration of larger DNA fragments, making them ideal for more
complex genetic studies.
The advent of recombinant DNA technology revolutionized the development in biology, and has offered new ~ 19 ~
International Journal of Veterinary Sciences and Animal Husbandry opportunities for the production of therapeutic
products. Recombinant DNA technology uses a vector as a tool, which is used as a vehicle to artificially carry genetic
material into host cells. Vectors have essential features that include origin of replication, presence of multiple cloning
sites and selectable marker. Generally, there are two types of vectors: cloning vectors and expression vectors, both of
which have similarities and differences with each other. All vectors had been created for different purposes in
molecular biology. Therefore, all useful cloning vectors and expression vectors should clearly be described so as to
use them accordingly.

9. Reference:
1. Google. (n.d.). [Plasmids and nucleoid]. Retrieved [december 21, 2024], from
https://www.slideshare.net/slideshow/plasmids-nucleoid/120165786
2. Google. (n.d.). [cloning Vectors]. Retrieved [december 21, 2024], from
https://www.aun.edu.eg/molecular_biology_institute/sites/default/files/Facilities/Procedure%20fro
m%20gene%20to%20protein/1-%20Cloning%20vectors.pdf
3. Google. (n.d.). [The Biology of Plasmids(book) By David Summers]. Retrieved [december 21,2024], from
https://books.google.com.pk/bookshl=en&lr=&id=a4lrPKQWjtAC&oi=fnd&pg=PR5&dq=plasmids&ots
=gfqZUCVEdS&sig=JSCZI-z1wbkcm7Q0liMkl_agPM4&redir_esc=y#v=onepage&q=plasmids&f=false
4. Google. (n.d.). [Yeast Artificial Chromosome (YAC)]. Retrieved [december 21, 2024],
fromhttps://www.slideshare.net/slideshow/yeast-artificial-chromosome/44965634?from_search=0
5. Google. (n.d.). [Expression vector]. Retrieved [december 21, 2024], from
https://www.slideshare.net/slideshow/expression-vector/100257337?from_search=1
6. Google. (n.d.). [TRANSCRIPTION AND TRANSCRIPTION FACTORS]. Retrieved [december 21, 2024], from
https://www.slideshare.net/ckoppala/transcription-and-transcription-factors?from_search=0
7. Google. (n.d.). [bacterial artificial chromosomes]. Retrieved [december 21, 2024], from
https://littleflowercollege.edu.in/upload/e_contents/files/944e4d2077e5badb287be51ba975116a.p df
8. Google. (n.d.). [viral vector]. Retrieved [december 21, 2024], from
https://www.ehs.ufl.edu/wp-content/uploads/2021/06/Viral-Vector-Fact-Sheet_210428-2.pdf 9. Google.
(n.d.). [ethical issues of gene cloning]. Retrieved [december 21, 2024], from
https://gurunanakcollege.edu.in/files/science/HUMAN-CLONING.pdf
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