Vector- Definition, Features, Types, Examples, Applications, Limitations

Vectors are substances that can carry foreign genetic material into another cell, where it can be replicated and/or expressed. Vectors are widely used in molecular biology and genetic engineering for various purposes, such as cloning, sequencing, expression, and gene therapy.

Vectors are usually DNA molecules, such as plasmids or viruses, that have been modified to contain specific sequences that allow them to function as vehicles for gene transfer. Vectors can also be RNA molecules, such as ribozymes or aptamers, that can bind to target molecules and modulate their activity.

The main features of vectors are:

They have an origin of replication that enables them to replicate independently of the host cell`s genome.
They have a selectable marker that confers a trait, such as antibiotic resistance or enzyme production, that allows the identification and selection of cells that contain the vector.
They have a multiple cloning site or a polylinker that contains several restriction enzyme recognition sites that facilitate the insertion of foreign DNA fragments into the vector.
They have a promoter sequence that drives the transcription of the foreign DNA in the host cell.
They have a termination sequence that signals the end of transcription.

Depending on the type and purpose of the vector, it may also have additional features, such as:

A signal sequence that directs the secretion of the foreign protein outside the cell.
A fusion tag that facilitates the purification or detection of the foreign protein.
A reporter gene that encodes a protein that produces a visible or measurable signal, such as fluorescence or luminescence.
A regulatory element that controls the expression of the foreign gene in response to external stimuli, such as temperature or inducers.

Vectors can be classified into different types based on their origin, size, host range, and function. Some of the common types of vectors are:

Plasmid vectors: circular DNA molecules that can replicate in bacteria and sometimes in other organisms, such as yeast or plants. They can carry small to medium-sized DNA inserts (up to 20 kb). Examples: pBR322, pUC19, pBluescript.
Bacteriophage vectors: viruses that infect bacteria and integrate their DNA into the host genome or form circular episomes. They can carry medium to large-sized DNA inserts (up to 50 kb). Examples: λ phage, M13 phage, P1 phage.
Cosmid vectors: hybrid vectors that combine features of plasmids and bacteriophages. They have a plasmid origin of replication and a bacteriophage packaging signal (cos site) that allows them to be packaged into phage particles for efficient transduction. They can carry large-sized DNA inserts (up to 45 kb). Examples: pHC79, SuperCos1.
Bacterial artificial chromosome (BAC) vectors: derived from bacterial F plasmids that can replicate stably in low copy numbers in bacteria. They can carry very large-sized DNA inserts (up to 300 kb). Examples: pBAC108L, pBeloBAC11.
Yeast artificial chromosome (YAC) vectors: derived from yeast telomeres, centromeres, and origins of replication that can replicate as linear chromosomes in yeast cells. They can carry extremely large-sized DNA inserts (up to 1000 kb). Examples: pYAC4, pYACneo.

Discussion of the characteristics and features of vectors

Vectors are DNA molecules that are used as vehicles to transfer foreign genetic material into another cell, where it can be replicated and/or expressed. Vectors have certain characteristics and features that enable them to perform this function effectively and efficiently. Some of the general characteristics of vectors are:

Origin of replication (ori): This is a DNA sequence that allows the vector to replicate autonomously inside the host cell, using the host`s replication machinery. The ori determines the copy number of the vector, which affects the yield and stability of the recombinant DNA.
Restriction sites: These are specific DNA sequences that can be recognized and cut by restriction enzymes, which are used to insert foreign DNA into the vector. Vectors should have compatible restriction sites for the DNA fragment of interest, and preferably multiple cloning sites (MCS) that contain a series of different restriction sites in a short region. This increases the flexibility and convenience of cloning different DNA fragments into the vector.
Selectable marker: This is a gene that confers a trait to the host cell that allows it to survive or grow under certain conditions, such as antibiotic resistance or enzyme production. Selectable markers are used to screen for the host cells that have received the vector, and to eliminate those that have not. Some vectors may also have reporter genes that produce a visible signal, such as color or fluorescence, to indicate the presence of the vector or the expression of the foreign DNA.
Size: The size of the vector affects its ability to carry foreign DNA and to be introduced into the host cell. Ideally, vectors should be small enough to be easily manipulated and isolated, but large enough to accommodate the desired DNA fragment. The size of the vector also influences its stability and maintenance in the host cell, as larger vectors may be more prone to recombination or deletion events.
Host range: This refers to the range of organisms or cell types that can be infected or transformed by the vector. Vectors should have elements that allow them to enter and replicate in the host cell of choice, such as viral capsids, bacterial pili, or transfection agents. Some vectors may also have elements that allow them to shuttle between different hosts, such as bacterial and eukaryotic cells, which can facilitate cloning and expression studies.
Integration: This refers to the ability of the vector to integrate into the host genome or to remain as an extrachromosomal element. Integration may be desirable for stable and long-term expression of foreign DNA, but it may also cause unwanted mutations or gene disruptions in the host genome. Extrachromosomal vectors may be easier to isolate and manipulate, but they may also be lost or diluted during cell division or growth.

Overview of the different 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.

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 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 E. coli as the host.

b. 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.

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 hybrid structure of cosmid enables the phage heads to be incorporated within all donor DNA for transfer.

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 pHC79 which is a cos-containing derivative of the vector pBR322.

c. Bacteriophage vector

Bacteriophage vectors are viruses that only infect bacteria and transform them efficiently while carrying large inserts.

Bacteriophages or phages have higher transformation efficiencies which increase the chances of recovering a clone containing the recombinant DNA segments.

The most important feature of a phage is the packaging system which enables the incorporation of large eukaryotic genes and their regulatory elements.

The use of phages also facilitates the isolation of larger quantities of DNA that can be used for the analysis of the insert.

Even though there are a number of phages that can and have been used as vectors, phage λ is the most convenient cloning vector.

It can selectively package a chromosome about 50 kb in length, and the size of the phage can be adjusted by removing the central part of the genome as it is not necessary for replication or the packaging of the donor DNA.

The use of a bacteriophage vector that can incorporate larger DNA segments decreases the number of clones required to obtain a particular DNA library with the entire genome of the organism.

Phage vectors are also effective as cloning vectors as the recombinant molecules formed after the cloning process are packaged into infective particles that can then be stored or handle efficiently.

Some of the common phages used as vectors include M13 phages, λ phages, and P1 phages.

Description of specific examples of vectors such as pBR322, pUC19, and λ phage

In this section, we will describe some of the specific examples of cloning vectors that are commonly used for different purposes. These include plasmid vectors, such as pBR322 and pUC19, and bacteriophage vector, such as λ phage.

Plasmid vectors

Plasmids are small circular DNA molecules that can replicate independently of the host chromosome in bacteria and other prokaryotes. They often carry genes that confer some advantage to the host cell, such as antibiotic resistance or metabolic capabilities. Plasmids can be manipulated in the laboratory to insert foreign DNA fragments and introduce them into bacterial cells by a process called transformation. Plasmids are widely used as cloning vectors because they are easy to isolate, purify, and manipulate, and they have high transformation efficiency and copy number.

pBR322

pBR322 is one of the first plasmids widely used as a cloning vector. It was derived from a naturally occurring plasmid called pMB1 that was isolated from E. coli. pBR322 has a size of 4361 base pairs (bp) and contains two antibiotic resistance genes: ampicillin resistance (ApR) and tetracycline resistance (TcR). These genes serve as selectable markers that allow the identification of bacterial cells that have taken up the plasmid. pBR322 also has an origin of replication (ori) from pMB1 that enables its autonomous replication in E. coli.

pBR322 has 21 unique restriction enzyme recognition sites, 11 of which are located within the ApR and TcR genes. These sites can be used to cut open the plasmid and insert foreign DNA fragments by molecular ligation. The insertion of foreign DNA into the ApR or TcR gene disrupts its function, resulting in the loss of antibiotic resistance. This allows the screening of recombinant plasmids by using different combinations of antibiotics on the growth medium.

pBR322 has been used for general cloning purposes in E. coli and other similar prokaryotes; however, it has some limitations. For instance, it can only carry DNA inserts up to 10 kilobases (kb) in size, and it has a low copy number (15-20 copies per cell). Moreover, it may be lost in continuous culture in the absence of selective pressure.

pUC19

pUC19 is another example of a plasmid cloning vector that was designed to overcome some of the drawbacks of pBR322. It was derived from a plasmid called pUC18 that was itself derived from another plasmid called pBR322. pUC19 has a size of 2686 bp and contains a single antibiotic resistance gene: ampicillin resistance (ApR). It also has an origin of replication (ori) from pMB1 that enables its high copy number (500-700 copies per cell) in E. coli.

pUC19 has a 54 bp multiple cloning site (MCS) or polylinker that contains 13 different restriction enzyme recognition sites. The MCS is located within a gene that encodes for the N-terminal fragment of β-galactosidase, an enzyme that catalyzes the hydrolysis of lactose into glucose and galactose. This gene serves as a reporter gene that allows the detection of recombinant plasmids by a colorimetric assay.

The insertion of foreign DNA into the MCS disrupts the β-galactosidase gene, resulting in the loss of enzyme activity. This can be visualized by using a substrate called X-gal, which produces a blue color when cleaved by β-galactosidase. Bacterial cells that have taken up the plasmid form white colonies on a medium containing X-gal and ampicillin, whereas cells that do not have the plasmid form blue colonies.

pUC19 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. It can carry DNA inserts up to 15 kb in size and has a high transformation efficiency.

Bacteriophage vector

Bacteriophages are viruses that infect bacteria and transfer their genetic material into the host cell. They often have high specificity for certain bacterial strains or species and can transduce their own DNA or foreign DNA into the host genome. Bacteriophages can be manipulated in the laboratory to replace some of their non-essential genes with foreign DNA fragments and introduce them into bacterial cells by a process called transfection. Bacteriophages have higher transformation efficiencies than plasmids and can carry larger DNA inserts.

λ phage

λ phage is an example of a bacteriophage vector that infects E. coli. It is a double-stranded DNA virus that has a linear genome with cohesive ends (cos sites) at both termini. The cos sites are recognized by an enzyme called terminase that cleaves and packages the phage DNA into a protein head during viral assembly.

The λ phage genome consists of three regions: left arm, right arm, and central region. The left arm and right arm contain genes essential for phage replication, integration, excision, and lysis. The central region contains genes non-essential for phage growth, such as genes involved in lysogeny, recombination, regulation, and immunity.

The central region can be removed and replaced with foreign DNA fragments up to 25 kb in size without affecting phage viability. This results in recombinant λ phage vectors that can infect E. coli cells and deliver foreign DNA into them. The recombinant λ phage vectors can be propagated either lytically or lysogenically depending on the environmental conditions.

Lytic propagation involves the production of many viral particles that lyse the host cell and release new viruses into the environment. Lysogenic propagation involves the integration of the viral genome into the host chromosome as a prophage that remains dormant until induced by stress factors. The choice of propagation mode depends on the purpose of cloning: lytic propagation is preferred for generating large amounts of recombinant DNA, whereas lysogenic propagation is preferred for maintaining stable clones over long periods.

λ phage has been used for cloning purposes where large DNA fragments are required for genomic studies or gene expression analysis. It has a high transformation efficiency and can infect different strains or species of bacteria by using different receptor molecules on its tail fibers.

Explanation of the various applications of vectors in molecular biology and genetic engineering

Vectors are DNA molecules that are used as vehicles to transfer foreign genetic material into another cell, where it can be replicated and/or expressed. Vectors have been developed and adapted for a wide range of uses in molecular biology and genetic engineering. Two primary uses are: (1) to isolate, identify, and archive fragments of a larger genome (2) to selectively express proteins encoded by specific genes.

Some of the major applications of vectors in molecular biology are:

Cloning: Vectors are the basic tools for cloning procedures, where a desired DNA fragment is inserted into a vector and then introduced into a host cell for replication. Cloning vectors allow the isolation and amplification of a particular gene or DNA segment from a complex genome. Cloning vectors can also be used to create DNA libraries, which are collections of clones containing different DNA fragments from a source organism.
DNA sequencing: Vectors can be used to sequence the nucleotide order of a DNA fragment by inserting it into a vector and then using specific enzymes or methods to determine the sequence. DNA sequencing can reveal the structure and function of genes, as well as their evolutionary relationships. Vectors can also be used to sequence entire genomes of organisms by using shotgun sequencing or next-generation sequencing techniques.
Gene expression: Vectors can be used to express a cloned gene in a host cell or organism, where the gene product (usually a protein) can be studied or harvested. Gene expression vectors contain regulatory elements that control the transcription and translation of the inserted gene. Gene expression vectors can be used for various purposes, such as producing recombinant proteins, studying gene regulation, creating transgenic organisms, or developing gene therapies.
Gene transfer: Vectors can be used to transfer genes between different cells or organisms, either horizontally or vertically. Horizontal gene transfer involves the transfer of genes between unrelated organisms, which can result in genetic diversity and adaptation. Vertical gene transfer involves the transfer of genes from parents to offspring, which can result in inheritance and evolution. Gene transfer vectors can be used for various purposes, such as creating hybrid organisms, introducing foreign genes into target cells, or modifying existing genes in situ.
Phage therapy: Vectors based on bacteriophages (viruses that infect bacteria) can be used as alternative agents for treating bacterial infections. Phage therapy involves the use of phage vectors that selectively target and kill pathogenic bacteria without harming beneficial bacteria or human cells. Phage therapy can overcome some of the limitations of antibiotics, such as resistance, toxicity, and side effects. Phage therapy can also be used for biocontrol, bioremediation, and biosensing applications.

Limitations of Vectors

Even though vectors have numerous applications in molecular biology and genetic engineering, they also have some limitations and challenges associated with their use. The following are some of the limitations of vectors:

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.
Overexpression of a particular type of genes in the host cell is a common problem associated with the use of vectors. This can result in toxicity, stress, or metabolic imbalance in the host cell.
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. For example, some vectors might not be compatible with each other or with the host cell.
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. The design, construction, and optimization of vectors require advanced techniques and expertise.
Some vectors are designed for transcription only, for example for in vitro mRNA production. These vectors are called transcription vectors. They may lack the sequences necessary for polyadenylation and termination, therefore may not be used for protein production.
Some vectors may introduce unwanted sequences or mutations into the host genome, which can affect the function or expression of the target gene or other genes. This can also cause ethical or safety concerns, especially when using viral vectors or human artificial chromosomes.
Some vectors may have low efficiency or specificity in transferring the recombinant DNA into the host cell. This can result in low yield or quality of the desired product or outcome.

Discussion of the limitations and challenges associated with using vectors.

In this article, we have discussed the definition, features, types, examples, applications, and limitations of vectors as substances that carry genetic material and introduce it into new cells. Vectors are essential tools for molecular biology and genetic engineering, as they enable the cloning, expression, and manipulation of different genes and proteins. However, vectors also have some challenges and drawbacks that need to be considered before choosing a suitable vector for a specific purpose.

Some of the limitations of vectors are:

Vectors may not be stable in different hosts or conditions, and may lose their function or integrity over time.
Vectors may not be able to carry large or complex DNA inserts, or may have low efficiency or specificity in transferring them into the host cells.
Vectors may cause unwanted effects on the host cells or tissues, such as toxicity, immunogenicity, insertional mutagenesis, or gene silencing.
Vectors may not be able to produce realistic or detailed images, such as photographs, textures, or shadows, as they are more suitable for flat, geometric, or abstract designs.

Therefore, it is important to select a vector that matches the requirements and objectives of the experiment or project. Different types of vectors have different advantages and disadvantages that need to be weighed carefully. Moreover, it is also important to optimize the vector design and delivery methods to ensure the safety and efficacy of the vector-mediated gene transfer.