UNIVERSITY OF CAPE COAST

COLLEGE OF AGRICULTURE

SCHOOL OF PHYSICAL SCIENCE

DEPARTMENT OF LABORATORY TECHNOLOGY

COURSE TITLE: BIOTECHNOLOGY II

COURSE CODE: LAT404A

DATE OF SUBMISSION:18TH JUNE 2024

ASSIGNMENT 2

1. Write on Vectors under the following headings: Features, Types, Examples, and Uses.
 ANSWER

Introduction

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 serve as carriers to transfer genetic material from one cell to another for various
purposes such as replication, expression, or isolation. They are used in molecular cloning
procedures to insert desired DNA into a host cell. The DNA transmitted by a vector is called
recombinant DNA, and the process is known as recombinant DNA technology. Vectors are
typically DNA sequences containing different parts with various functions. They usually consist
of an insert, also known as a transgene, carrying the recombinant DNA, and a larger sequence
known as the vector backbone, which determines the vector's structure. Vectors can be classified
into different types based on various characteristics, and the selection of vectors depends on the
specific purpose of the process. They are a crucial component of genetic engineering, facilitating
the transfer of DNA fragments from one cell to another. Vectors possess unique features that
carry gene sequences, enabling them to survive within the host cell.

The gene transfer process also differs in different vectors where some enter the host cell and get
incorporated into the host DNA. In contrast, 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.

Vectors can be reused for multiple processes as these can be recovered at the end of the process.

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.
Vector. Created with BioRender.com.

CHARACTERISTICS OR FEATURES OF VECTORS

The following are some of the characteristic features of vectors;

 Vectors must be able to replicate independently, which relies on specific sequences

enabling them to commence replication and transmission within the host cell. Some
vectors also contain sequences that allow to production of essential proteins for the
inserted DNA, regulation of the process, and transfer of the inserted DNA between
different vectors.
 An ideal vector should be small enough to be easily integrated into the host genome. Its
small size also allows for the transfer of large inserts.
 Vectors should be easily isolated and purified for reuse in multiple processes.
  Effective vectors should include components that help determine if the host cell has
received the vector, such as genes providing resistance to antibiotics or producing
specific proteins, known as marker genes.
 Many vectors require unique restriction enzyme recognition sites for the insertion of
vector DNA, and some are designed with multiple cloning sites for various restriction
enzymes.
 The introduction of vectors into the host cell should be uncomplicated, depending on
several factors.
 In gene transfer processes, vectors must be capable of integrating themselves or the
recombinant DNA into the genome of the host cell.
 The introduction of recombinant DNA into the vector must not disrupt the vector's
replication cycle.

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 groups of vectors
that are used for different purposes;

1. Cloning vectors

Cloning vectors are vectors that can replicate 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.

Cloning vectors are of further different types that are defined by different features unique to each
type of vector.
a. Plasmid vector
Plasmids are small circular DNA molecules that can replicate autonomously within a host
cell, making them a popular choice in recombinant DNA technology. They are commonly
used in bacteria and yeasts due to their small size, which allows for the separation of
recombinant DNA from the host's genomic DNA. The size of plasmids can range from a
few thousand base pairs to over 100 kilobases, but it affects the maximum size of insert
DNA it can carry. Plasmids can carry insert DNA less than 20 kb due to decreased
cloning efficiency and stability. Bacterial plasmids contain ori sequences that control
plasmid replication and determine the possibility of two plasmids coexisting within the
same host cell.

b. Cosmid
Cosmid vectors are hybrid vectors made up of plasmid and phage λ vectors, capable of
incorporating up to 42 kb of DNA. They are large-sized vectors with sizes ranging from
400 base pairs to 30 kb and can carry DNA sequences ranging from 28 to 46 kb. They can
 replicate within the host cell like plasmids or remain packaged like a phage. The hybrid
structure allows phage heads to be incorporated within donor DNA for transfer. The use
and production of cosmid vectors have increased due to their efficiency and selective
recovery of larger hybrids.

c. Bacteriophage vector

Bacteriophage

Bacteriophage vectors are viruses that efficiently infect bacteria and transform them with large
inserts. They have higher transformation efficiencies, increasing the chances of recovering a
clone containing recombinant DNA segments. Phage vectors have a packaging system that
allows for the incorporation of large eukaryotic genes and regulatory elements. They facilitate
the isolation of larger quantities of DNA for insert analysis. Phage λ is the most convenient
cloning vector, as it can selectively package a 50 kb chromosome. This reduces the number of
clones needed to obtain a specific DNA library. Common phages used as vectors include M13, λ,
and P1 phages.

c. Bacterial artificial chromosome

Bacterial artificial chromosomes (BACs) are DNA molecules used to clone DNA segments in
bacteria cells, such as E. coli. They have a bacteria-derived F-factor replication origin, allowing
for the propagation of large DNA fragments in a supercoiled circular form. BACs are considered
superior over yeast and mammalian artificial chromosomes due to their F-factor, which reduces
insert chimerism and instability. They can insert DNA segments as large as 300,000 base pairs,
reducing the number of clones and cycles needed. BAC libraries are used for positional cloning,
physical mapping, and genome sequencing. However, BACs can result in unpredicted
expression.

d. Yeast artificial chromosome

Yeast artificial chromosomes (YACs) are engineered DNA molecules used to clone DNA inserts
within yeast cells, particularly Saccharomyces cerevisiae. These cloning vectors can clone up to
500 kb of DNA, significantly higher than traditional methods. YACs are also useful in DNA
sequencing and analysis and can clone complete sequences of larger genomes. They can be used
for unstable sequences in prokaryotic systems but introduce high levels of chimerism and insert
rearrangement. YACs are difficult to handle and have lower efficiencies compared to bacterial
artificial chromosomes. pYAC4, a commonly used cloning vector, is one of the most used yeast
artificial chromosomes.

e. Human artificial chromosome

Human artificial chromosomes (HACs) are extrachromosomal DNA fragments that act as new
chromosomes within the human cell. They offer long-term stable maintenance and no upper limit
on the DNA insert size, allowing for natural gene expression mimicking. However, HACs have
limited applications due to technical difficulties during gene loading and ill-defined vector
structures. Advances in genetic engineering have increased the use of HACs.

2. Viral vectors
Viral vectors are one of the most effective means of gene transfer to modify host cells or tissues
and manipulate them to express different types of genes.

The concept of using viruses as vectors arose from the fact that viruses are very effective in
transducing their genetic information into the host cell.

During viral transduction, the non-essential viral genes are replaced with foreign DNA sequences
of therapeutic interest to produce recombinant viral vectors.

Currently, different groups of viruses have been studied doe their possible use as viral vectors to
deliver genes to provide transient or permanent transgene expression.

The use of viral vectors also enables location specificity with unique injection technology within
a specific period.

Some of the common virus groups considered for viral vectors are adenoviruses, retroviruses,
poxviruses, and adeno-associated viruses.

The choice of a particular virus as a vector depends on several factors including efficiency of
transgenic expression, ease of production, safety, and stability.

Different clinical trials have been held with different potential viral vectors that are suitable for
different purposes.

Adenoviruses have been used for the transfer of tumour suppressor genes in cancer treatment,
and retroviruses are studied for their potential use in tissue repair and engineering.
3. Expression vector

Expression vectors are vectors that enable the expression of cloned genes 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 the 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.

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 and are introduced into the host cells to
code for mRNAs.

The expression vectors are often used to produce 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 that carry origins of replication from two different hosts, which enables them to
‘shuttle’ between the two hosts.

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 enables the recovery and
maintenance of these vectors in bacterial cells.

Three different shuttle vectors depend on the type of replication system utilized by the vectors.

Transiently replicating shuttle vectors that need to be recognized by large T antigens 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 cell types for gene
expression.
5. Secretion vector

Secretion vectors are a type of specialized expression vector that expresses the cloned genes to
produce proteins at locations other than the cytoplasm.

The transport of protein products 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 vectors has many advantages including higher yield, a 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 using 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.

EXAMPLES OF VECTORS

The following are some of the examples of vectors that are commonly used for different genetic
engineering processes;

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 of 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 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 are 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.

pUC19

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 colour 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.
The colony screening after cloning with pUC19 is due to the presence of a selective marker that
encodes for the N-terminal fragment of β-galactosidase.

λ phage

λ-phage is an example of a bacteriophage that infects the bacterial species, Escherichia coli (E.
coli).

This vector is more effective than other plasmid vectors as it has a higher efficiency in entering
bacterial cells to incorporate the recombinant DNA within the host genome.

It is a double-stranded DNA bacteriophage that contains an ori sequence required for replication
and several DNA sequences encoding regulatory and replicative proteins.

The phage DNA replicates by the combination of theta and rolling circle replication process to
produce a linear dsDNA. It is then followed by the cos sequence, which enables the
circularization of the genome after infection.

The DNA sequences between the two arms of the vector are not essential which are then
replaced with the recombinant DNA during cloning.

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 uses or 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
proteins 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.
REFERENCES

Sapkota, A. (2024, May 25). Vector: Features, Types, Examples, Uses, Diagram. Microbe Notes.

Retrieved from https://microbenotes.com/vector-molecular-biology/

Asami, Junko et al. Bacterial artificial chromosomes as an analytical basis for gene
transcriptional machinery. Transgenic research vol. 20,4 (2011): 913-24. doi:10.1007/s11248-
010-9469-3