VECTORS (nature, uses, types:

Bacteriophase, cosmid,
plasmid, BAC, YAC)
Introduction
A major step in genetic engineering is the manipulation of DNA for cloning purposes.
Cloning allows the genetic engineer to isolate genes of interest away from their host genomes
and insert them into carrier molecules where they can be more easily manipulated or
otherwise studied. To accomplish this, genes or other genetic elements to be cloned are
inserted into cloning vectors that replicate in a host organism.
Definition:
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.
• Vectors are used as a tool in molecular cloning procedures so as to introduce the desired
DNA insert into a host cell.
• The DNA insert that is transmitted by a vector is termed recombinant DNA, and the process
is also known as recombinant DNA technology.
• Usually, the vectors are DNA sequences that carry different parts involved in
different functions. 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 can be classified into different types depending on different characteristics.
The selection of vectors thus depends on the purpose of the process.
• Vectors are an important component of the genetic engineering process as these
form the basis for the transfer of DNA fragments from one cell to another.
• 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.
• Even though vectors are usually DNA sequences, viruses and other particles can also
function as vectors in processes like transduction
• A cloning vector is a category of vectors that are essential for cloning procedures.
These vectors have different sequences that enable them to initiate replication in
host cells as well as propagate within the host.
Characteristics of vectors
• Vectors should be capable of replicating autonomously, which, in turn, depends on
the presence of particular sequences in the vector that enables them to initiate
replication and propagation within the host cell. Some vectors might even have
sequences that allow the production of proteins essential for the inserted DNA,
regulation of the process, and further transfer of the insert between different
vectors.
• The size of an ideal vector should also be small enough for it to be incorporated into
the host genome. The small size of the vector also enables it to incorporate a large-
sized insert for transfer.
• Vectors should be easy to isolate and purify as these need to be recovered and reused for
multiple processes.
• For a vector to be effective, these should also have certain components that facilitate the
process of determining whether the host cell has received the vector.
• Most vectors used in this process have a gene that either provides resistance to an antibiotic
or produces a particular type of protein. These components are called marker genes.
• Many vectors also require unique restriction enzyme recognition sites that enable the
insertion of the vector DNA in the presence of specific restriction enzymes.
• However, many vectors have been designed with a series of restriction sites close to multiple
cloning sites that increases the possible restriction enzymes that can be used to digest the
sequence.
• The introduction of vectors into the host cell should be easy, which depends on a number of
factors.
• In the case of gene transfer processes, it is important that the vector is capable of integrating
itself or the recombinant DNA into the genome of the host cell.
• It is important that the introduction of recombinant DNA into the vector doesn’t affect the
replication cycle of the vector.
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.
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 genetically modified
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. 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.
7. Recombinant DNA technology has been used in clinical microbiology in different
approaches like recombinant antigens, recombinant vaccines, and diagnostic probes.
8. Recombinant antigens prepared by cloning techniques by using cloning vectors have
been used for the screening of diseases like HIV, HCV, and CMV.
9. Vectors are one of the components in molecular biology which enable numerous
studies related to cell structure, nucleic acid composition, and genetic engineering
techniques.
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
• There are four major types of vectors: plasmids, bacteriophages and other viruses,
cosmids, and artificial chromosomes
• Each type has its own advantages and applications, so the selection of the proper
cloning vector is critical to the success of any cloning experiment.
• Most cloning vectors share three important features: an origin of replication; a region
of DNA that bears unique restriction sites, called a multicloning site or polylinker; and
a selectable marker.
i) Plasmid vector
• Plasmids are small extrachromosomal circular DNA molecules capable of replicating
autonomously within the host cell.
• These are also termed as the workhorse cloning vector in recombinant DNA technology.
Plasmids are widely used as vectors in all three domains of life; however, these are
frequently used in bacteria and yeasts.
• The most important feature of plasmids that makes them one of the best vectors is their
small size. The small size of the plasmid facilitates the separation of recombinant DNA
from the host’s genomic DNA.
• The size of plasmids ranges from a few thousand base pairs to more than 100 kilobases.
The small size of the vector does, however, affect the maximum size of the insert DNA it
can carry.
• Plasmids can carry insert DNA that is less than 20 kb as the cloning efficiency and
plasmid stability decrease with the size of the vectors.
• The autonomous replication of plasmid is made possible by the presence of genes and
sequences that can initiate plasmid replication independent of the host’s replication cycle.
• Bacterial plasmids contain ori sequences that not only control plasmid replication but also
determine the possibility of two plasmids coexisting within the same host cell.
• Different plasmids have different types of selective markers, but the most common
markers include antibiotic resistance and the production of the β-galactosidase enzyme.
• Some of the most widely used plasmids are pBR322, pUC, and pBluescript vectors that use
[Link] as the host
• pUC19, an E. coli plasmid, has an ori that generates a “high copy number” because it
directs about 50–100 plasmid replications in the course of one cell cycle. High copy number
is often important because it facilitates plasmid purification and can dramatically increase
the amount of cloned gene product produced by the cell.
• Some plasmids have two origins of replication, each recognized by different host
organisms. These plasmids are called shuttle vectors because they can be transferred, or
“shuttle,” from one host to another. YEp24 is a shuttle vector that can replicate in yeast
(Saccharomyces cerevisiae) and in E. coli because it has the 2µ circle yeast replication
element and the E. coli origin of replication
Selectable Marker
• Following the uptake of vector by host cells, it is necessary to discriminate between
cells that successfully obtained vector (transformants) from those that did not (non-
transformants).
• Furthermore, selective conditions for the presence of plasmid must be maintained,
otherwise the host cell may stop replicating it.
• This is achieved by the presence of a gene encoding a protein needed for the cell to
survive under certain conditions. Such a gene is called a selectable marker. In the
case of pUC19, the selectable marker, ampR, encodes the ampicillin-resistance
enzyme.
• Because E. coli is normally susceptible to ampicillin, only those cells that have taken
up plasmid (i.e., transformants) will grow when plated on agar containing ampicillin
Multicloning Site or Polylinker
• In order to clone a fragment of DNA into a plasmid, both the fragment and plasmid
are cut with the same restriction enzyme (or enzymes) so that compatible sticky ends
are generated. It is essential that these restriction enzymes cut at only one place in
the plasmid.
• Cleavage at a unique restriction site generates a linear plasmid. Alternatively, two
different, unique sites may be cleaved and the DNA sequence between the two sites
replaced with cloned DNA. Plasmids used for cloning have been designed with many
restriction sites clustered in a single region called the multicloning site (MCS)
ii) Phase vector:
• Phage vectors are phage genomes that have been genetically modified to include useful
restriction enzyme recognition sites for the insertion of foreign DNA. Once DNA has
been inserted, the recombinant phage genome is packaged into viral capsids and used
to infect host cells.
• The resulting phage lysate consists of thousands of phage particles that carry cloned
DNA as well as the genes needed for host lysis. Two commonly used vectors are derived
from the E. coli bacteriophages T7 and lambda (ƛ), both of which have double-stranded
DNA genomes
• Bacteriophage lambda is a useful cloning vector because its biology is well understood,
it can hold larger amounts of DNA than most plasmids, and DNA can be efficiently
packaged into phage particles in vitro.
• Phage lambda has a large number of genes; however, a third of the lambda genome
is not essential for infectivity and can be replaced with foreign DNA. This allows
relatively large DNA fragments, up to about 20 kbp, to be cloned into lambda.
• To facilitate the use of lambda as a molecular cloning vector, many of its restriction
enzyme sites have been altered and a multiple cloning site (MCS) containing the
gene for β-galactosidase has been added to select for recombinant vectors.
iii) Cosmid:
• Cosmid vectors are hybrid vectors composed of plasmid and phage λ vectors, capable of
incorporating up to 42 kb of DNA.
• Cosmid vectors are prepared by the insertion of the cos region of the phage vector into
the plasmid vectors.
• These engineered vectors have a cos site from phage and a selectable marker, origin of
replication, and MCS from plasmids (thus the term “cos-mid”).
• These hybrid vectors replicate as plasmids within the host cell, but the presence of the
cos sites means that the vector can be packaged into phage capsids and transferred to
new host cells by transduction.
• Cosmid vectors are large-sized vectors with sizes ranging from 400 base pairs to 30 kb.
These can carry DNA sequences having sizes ranging from 28 to 46 kb.
• Cosmid vectors are created in order to incorporate large-sized DNA molecules that cannot
be carried by plasmids.
• Since these are hybrid vectors, these can replicate within the host cell like plasmids or
remain packaged like a phage.
• Cosmid vectors do not have many phage characteristics except the signal sequences that
promote phage-head stuffing.
• The use and production of cosmid vectors have increased over the years as the packaged
system is highly efficient and selective for the recovery of larger hybrids.
• One of the examples of the cosmid vectors prepared and used in practice are cosmid
pHC79which is a cos-containing derivative of the vector pBR322.
iv) Artificial Chromosomes
• Artificial chromosomes are special cloning vectors used when particularly large
fragments of DNA must be cloned, as when constructing a genomic library.
• In fact, the genomic library containing the human genome was constructed in
bacterial artificial chromosomes (BACs), which then enabled nucleotide sequencing.
BACs were also used in the construction of a synthetic microbial genome.
• Like natural chromosomes, artificial chromosomes replicate only once per cell cycle.
Yeast artificial chromosomes (YACs) were developed first and consist of a yeast
telomere (TEL) at each end, a centromere sequence (CEN), a yeast origin of
replication (ARS, autonomously replicating sequence), a selectable marker such as
URA3, and an MCS to facilitate the insertion of foreign DNA.
• YACs are used when extraordinarily large DNA pieces (up to 1,000 kb) are to be cloned.
• BACs were developed, in part, because YACs tend to be unstable and may recombine
with host chromosomes, causing mutations and rearrangement of the cloned DNA.
• Although BACs accept smaller DNA inserts than do YACs (up to 300 kb), they are
generally more stable. BACs are based on the F fertility factor of E. coli
• The example shown in figure is typical in that it includes genes that ensure a replication
complex will be formed (repE), as well as proper partitioning of one newly replicated BAC
to each daughter cell (sopA, sopB, and sopC).
• It also includes features common to many plasmids such as an MCS within the lacZ gene
for blue/white colony screening and a selectable marker, in this case for resistance to the
antibiotic chloramphenicol (CmR).
Bacterial artificial chromosome
• Bacterial artificial chromosomes are engineered DNA molecules that are used to clone
DNA segments in bacteria cells (usually E. coli).
• These consist of a bacteria-derived F-factor replication origin which enables the
propagation of large DNA fragments in a supercoiled circular form.
• Bacterial artificial chromosomes can carry a much larger size of insert DNA as
compared to plasmid or phage vectors.
• These vectors are considered superior over other artificial chromosomes like yeast
artificial chromosomes, and mammalian artificial chromosomes as the F-factor found
in the bacteria reduces insert chimerism and instability that might arise during the
process.
• These are highly efficient as DNA segments as large as 300,000 base pairs can be
inserted into bacterial artificial chromosomes, which decreases the number of clones
and cycles to be performed to obtain the desired result.
• BAC libraries have been used to generate large genomic DNA inserts for processes like
positional cloning, physical mapping, and genome sequencing.
• BAC cloning system has been increasingly used in genetic engineering due to its stability
and ease of use as compared to other similar vectors.
• However, BACs have been associated with the random insertion of DNA fragments into
the host genome resulting in unpredicted expression.
Yeast artificial chromosomes
• Yeast artificial chromosomes are engineered DNA molecules that are used to clone DNA
inserts within the yeast cells, particularly Saccharomyces cerevisiae.
• YACs have been developed in order to clone large sequences of DNA so as to increase the
efficiency of the process.
• YACs can clone up to 500 kb of DNA, which is much higher than most traditional cloning
vectors.
• Even though these are frequently used as cloning vectors, they are also helpful in other
genetic processes like DNA sequencing and analysis.
• These are also unique in their ability to clone the complete sequences of larger
genomes that exceed the limits of traditional techniques.
• Since yeast cells are eukaryotic cells, YACs can be used for unstable sequences when
cloned in prokaryotic systems
• 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.
• 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 yeast artificial chromosomes have been created over the years that are
then used for different purposes.
• One of the most commonly used examples of yeast artificial chromosomes includes
pYAC4,which has been extensively used as a cloning vector.