ABT 301 Plant Biotechnology (2+1)

Name: P.MOHAMED SIDDIK

I’d No: 2021031052

Cloning Vectors: Their Components and Types.

Introduction

Cloning vectors play a pivotal role in genetic engineering, facilitating the manipulation and
transfer of genes across different organisms. These vectors, essential tools in molecular biology.
Are carefully designed to carry foreign DNA into host cells. Let’s delve into their components
and explore the diverse types that scientists employ in genetic engineering endeavors.

Cloning vector

A cloning vector is also a fragment of DNA which is capable of self-replication and stable
maintenance inside the host organism, It can be extracted from a virus, plasmid or cells of a
higher organism. Most of the cloning vectors are genetically engineered. It is selected based
upon the size and the kind of DNA segment to be cloned.

Components of Cloning Vectors:

Origin of Replication (ori): This fundamental element allows the vector to replicate

Autonomously within a host ce4ll. Plasmids, a common type of cloning vector, often contain an
ori that dictates the initiation of replication.

Selectable Marker: These are genes that confer a survival advantage to host cells harboring the
vector. Common markers include antibiotic resistance genes, allowing scientists to selectively
grow only those cells that have taken up the vector.

Multiple Cloning Site (MCS): Also known as a polylinker, the MCS is a region with

Multiple unique restriction sites. It facilitates the insertion of foreign DNA by providing
numerous options for restriction enzymes to cut and integrate the desired genetic material.

Reporter Genes: These genes produce easily detectable traits, aiding researchers in identifying
cells that have successfully incorporated the vector. Examples include genes encoding for
fluorescent proteins or enzymes that catalyze colorimetric reactions.
Plasmid Backbone: The foundational structure of a cloning vector, the plasmid backbone
includes all components necessary for the vector's stability and replication. It encompasses the
ori, selectable marker, and often the MCS.

Types of Cloning Vectors:

Plasmids:

 Circular DNA molecules separate from the chromosomal DNA,

commonly used due to their ease of manipulation and replication.
 Plasmids were the first vectors to be used in gene cloning.
 They are naturally occurring and autonomously replicating extra-
chromosomal double- stranded circular DNA molecules. However, not
all plasmids are circular in origin.
 They are present in bacteria, archaea, and eukaryotes.
 The size of plasmids ranges from 1.0 kb to 250 kb.
 DNA insert of up to 10 kb can be cloned in the plasmids.
 The plasmids have high copy number which is useful for production of
greater yield of recombinant plasmid for subsequent experiments.
 The low copy number plasmids are exploited under certain conditions
like the cloned gene produces the protein which is toxic to the cells.
 Plasmids only encode those proteins which are essential for their own
replication.
  Examples: pBR322, pUC18, F plasmid, Col plasmid

Bacteriophages (Phage Vectors):

 Bacteriophages or phages are viruses which infect bacterial cells.

 The most common bacteriophages utilized in gene cloning are Phage 2
and M13 Phage.
 A maximum of 53 kb DNA can be packaged into the phage.
 If the vector DNA is too small, it cannot be packaged properly into the
phage.
 Examples: Phage Lambda, M13 Phage, etc.
 Viruses that infect bacteria can serve as vectors. They integrate their
DNA into the host genome, allowing the foreign DNA to replicate along
with the bacterial DNA.
Cosmid Vectors:

 Hybrid vectors combining features of plasmids and bacteriophages.

 They can carry larger DNA fragments than plasmids and maintain
stability during replication.
 They are capable of incorporating the bacteriophage A. DNA segment.
This DNA segment contains cohesive terminal sites (cos sites).
 Cos sites are necessary for efficient packaging of DNA into A phage
particles.
 Large DNA fragments of size varying from 25 to 45 kb can be cloned.
 They are also packaged into 2. This permits the foreign DNA fragment
or genes to be introduced into the host organism by the mechanism of
transduction.
 The pHV79 is one of the most common cosmids used in genetic
engineering.

Bacterial Artificial Chromosomes (BACs):

 Large vectors designed to carry extensive DNA inserts, mimicking the

behavior of natural bacterial chromosomes. BACs are crucial for
cloning large genomic fragments.
 Bacterial artificial chromosomes are similar to E. coli plasmid vectors.
 They contain ori and genes which encode ori binding proteins. These
proteins are critical for BAC replication.
 It is derived from naturally occurring F’plasmid.
  The DNA insert size varies between 150 to 350 kb.

Yeast Artificial Chromosomes (YACS):

 Employed for cloning even larger DNA fragments, YACs mimic yeast
chromosomes.
 They can accommodate DNA fragments up to several hundred
kilobases.
 A large DNA insert of up to 200 kb can be cloned.
 They are used for cloning inside eukaryotic cells. These act as
eukaryotic chromosomes inside the host eukaryotic cell.
 It possesses the yeast telomere at each end.
 A yeast centromere sequence (CEN) is present which allows proper
segregation during meiosis.
 The ori is bacterial in origin.
 Both yeast and bacterial cells can be used as hosts.
Retroviral Vectors:

 Derived from retroviruses, these vectors can integrate their genetic

material into the host genome.
 They are commonly used in gene therapy due to their ability to
achieve stable, long-term expression in host cells.
 Retroviruses are the virus with RNA as the genetic material.
 Retroviral vectors are used for introduction of novel or manipulated
genes into the animal or human cells.
 The viral RNA is converted into DNA with the help of reverse
transcriptase and henceforth, efficiently integrated into the host cell.
 Any gene of interest can be introduced into the retroviral genome. This
gene of interest can then integrate into host cell chromosome and
reside there.

Adenoviral Vectors:

 Based on adenoviruses, these vectors efficiently deliver genes into a

variety of cell types.
 They are popular in gene therapy and vaccine development due to
their capacity for transient expression without genomic integration.

Episomal Vectors:

 Unlike vectors that integrate into the host genome, episomal vectors
remain separate, often as extrachromosomal plasmids.
 This ensures that the foreign DNA doesn’t disrupt host genes and is
useful for certain applications.

Gateway Vectors:

 These vectors use the Gateway cloning technology, allowing for easy
and efficient transfer of DNA fragments between different vectors.
 It simplifies the process of constructing complex DNA sequences.

T-DNA Vectors:
  Widely used in plant genetic engineering, T-DNA (transfer DNA) vectors
are derived from the Ti plasmid of Agrobacterium tumefaciens.
 They transfer genes into plant cells, leading to the integration of
foreign DNA into the plant genome.

Suicide Vectors:

 These vectors contain a lethal gene that prevents the growth of host
cells that haven’t successfully incorporated the vector.
 They are useful for selecting only those cells that have undergone the
desired genetic modification.

Artificial Chromosomes:

 Beyond BACs and YACs, newer artificial chromosomes like Human

Artificial Chromosomes (HACs) have been developed. HACs are
designed to carry large DNA fragments and mimic natural
chromosomes more closely.
 Human artificial chromosomes are artificially synthesized.
 They are utilized for gene transfer or gene delivery into human cells.
 It can carry large amounts of DNA inserts.
 They are used extensively in expression studies and determining the
function of the human chromosomes.
CRISPR Vectors:

 With the advent of CRISPR-Cas technology, vectors have been adapted

to carry components of the CRISPR system for precise genome editing.
 These vectors enable targeted modification of specific genes in various
organisms

Integrating and Non-Integrating Vectors:

 Integrating vectors, like retroviral vectors, become part of the host

genome.
  Non-integrating vectors, such as adenoviral vectors, persist in the host
cell without integrating into the genome.

Understanding the strengths and limitations of these diverse vectors allows

scientists to choose the most suitable tool for their specific research or
therapeutic goals. As biotechnology evolves

The development of novel vector types continues to enhance the precision

and efficiency of genetic engineering techniques.

Specialized Cloning Vectors:

 Expression Vectors:
Engineered for the efficient expression of foreign genes in host cells.
They often contain additional elements such as promoters and
enhancers to drive high levels of gene expression.

 Shuttle Vectors:
Designed to move genes between different host organisms. These
vectors have compatible features for replication and expression in
multiple species.
 RNAI Vectors:
Specifically crafted for RNA interference studies. They carry sequences
that generate short interfering RNAs (siRNAs) or short hairpin RNAs
(shRNAs), allowing researchers to selectively silence target genes

Applications of Cloning Vectors:

 Recombinant DNA Technology: Cloning vectors are
fundamental in creating recombinant DNA molecules.This
process involves combining DNA from different sources, enabling
the study and manipulation of specific genes.
 Gene Therapy: Vectors play a crucial role in gene therapy by
delivering therapeutic genes into target cells. Modified viruses,
such as adenoviruses or lentiviruses, are often used as gene
delivery vectors in clinical applications.
  Functional Genomics: Cloning vectors contribute to
understanding gene function by allowing researchers to
introduce or modify genes in a controlled environment. This aids
in deciphering the role of specific genes in various biological
processes.
 Protein Production; Expression vectors are utilized for the
production of recombinant proteins. Host cells, often bacteria,
yeast, or maumalian cells, are engineered to perde large
quantities of a desired protein for research or therapeutic
purposes
 Transgenic Organism Creation: Cloning vectors are
instrumental in creating transgenic Organisms with foreign genes
integrated into their genomes. This has applications in
Agriculture, where crops can be engineered for improved traits,
and in research to study Gene function in whole organisms.

Features of Cloning Vectors

The cloning vectors possess the following features

 A cloning vector should possess an origin of replication so that it com

self-replicates the host cell.
 It should have a restriction site for the insertion of the target DNA.
 It should have a selectable marker with an antibiotic resistance gene
that facilitates screening of the recombinant organism.
 It should be small in size so that it can easily integrate into the host
cell,
 It should be capable of inserting a large segment of DNA.
 It should possess multiple cloning sites.
 It should be capable of working under the prokaryotic and eukaryotic
systems
Challenges in Cloning:

 Size Limitations: Some vectors have size restrictions on the DNA

they can carry. Larger DNA fragments may be challenging to clone
using certain vectors.
 Insert Stability: Maintaining the stability of the inserted DNA,
especially in rapidly dividing cells, is crucial. Unstable inserts may be
lost during cell division.
 Promoter Compatibility: Ensuring compatibility between the cloned
gene and the promoter in the vector is essential for proper gene
expression.
 As technology advances, researchers continually refine cloning vectors
and develop new ones to address specific research needs, pushing the
boundaries of genetic engineering and molecular biology.

Conclusion

The advent of recombinant DNA technology revolutionized the development

in biology, and has offered new 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. All vectors had been created for
different purposes in molecular biology. Therefore, all useful cloning vectors
should clearly be described so as to use them accordingly.

Reference

 Telkar S, Nalina M, Navale PM. Basic Concept of Biotechnology Editors

2015
 Marinda O. Molecular Biology: Gene cloning 2018, 1-24,
 https://www.sciencedirect.com/topics/agricultural-and-biological-
sciences/vector- molecular-biology
 https://nptel.ac.in/courses/102103017/pdf/lecture%2035.pdf