In this article we will discuss about Vectors:- 1. Definition of Vectors 2. Characteristic Features of an Ideal Vector 3. General Construction.
Definition of Vectors:
A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the gene of interest (a foreign genetic sequence) is integrated. When we insert a foreign genetic sequence into the vector the aim is either to obtain numerous copies of the gene of interest or to obtain the product of that.
Accordingly the design and features of the vectors used for the assigned activity vary. Due to this there are two types of vectors − the expression vectors and cloning vectors.
Characteristic Features of an Ideal Vector:
1. It should be able to replicate autonomously. When the objective of cloning is to obtain a large number of copies of the DNA insert, the vector replication must be under relaxed control so that it can generate multiple copies of itself in a single host cell.
2. It should be easy to isolate and purify.
3. It should be easily introduced into the host cells, i.e., transformation of the host with the vector should be easy.
4. The vector should have suitable marker genes that allow easy detection and/or selection of the transformed host cells.
5. When the objective is gene transfer, it should have the ability to integrate either itself or the DNA insert it carries into the genome of the host cell.
6. The cells transformed with the vector containing the DNA insert (recombinant DNA) should be identifiable and selectable from those transformed by the unaltered vector.
7. A vector should contain unique target sites for as many restriction enzymes as possible into which the DNA insert can be integrated.
8. When expression of the DNA insert is desired, the vector should contain at least suitable control elements, e.g., promoter, operator and ribosome binding sites.
General Construction of a Vector:
A DNA molecule should possess the following three essential characteristics to act as a cloning vector:
(a) Origin of Replication:
It is required for autonomous replication of the plasmid using the host’s replication machinery. Almost all commonly used plasmids are based on the ColE1 origin of replication (ori). Naturally occurring origins of replication are so designed that they always keep the copy number (number of similar copies of a DNA molecule) down (5-10 copies per cell).
This is done in order to reduce the load on the host’s DNA replication machinery. While a high copy number is disadvantageous in a natural system, it is a desirable feature in a cloning vector—since the whole idea of gene cloning is to easily isolate substantial quantities of a particular DNA sequence.
Therefore, considerable work has gone into engineering the ColE1 ori such that the negative regulatory mechanisms that limit the copy number of plasmids are disabled. Modern plasmid vectors are, therefore, often called ‘runaway replicons’ and are present at 100 to 1,000 copies per cell.
(b) Selectable Markers:
After the construction of recombinant DNA we insert it into a suitable host. When a host receives the recombinant DNA we call that it has been transformed. In a mixture of host cells all will never get transformed. So we must have a mean to differentiate between the transformed and non-transformed host cells. This is where selectable markers help us.
Selectable marker genes are used as tools to identify those cells which have successfully incorporated genes for a desired trait during transformation. Since the percentage of cells that will incorporate genetic material during transformation is low, selectable markers are extremely important because they act as “genetic tags” to easily identify successfully modified cells.
There are four categories of selectable marker genes presently used as tools to develop novel products.
The different types of selectable markers and their respective modes of action are described below.
1. Antibiotic Resistant Marker Genes:
Antibiotic resistant marker genes confer the trait of resistance to a specific antibiotic. For example, the neomycin phospho-transferase-II (NPT-II) gene, is a selectable marker gene that is the blueprint for resistance to the antibiotics neomycin and kanamycin.
While they are useful in the early development of transgenic cells, antibiotic resistant marker genes are engineered to be expressed at minimal and often undetectable levels in the final product.
2. Herbicide Tolerant Marker Genes:
Herbicide tolerant marker genes confer the trait of tolerance to the application of a specific herbicide. For instance, the gene conferring tolerance to the herbicide glufosinate ammonium is often used as a selectable marker in plant biotechnology. Those cells which survive the exposure to the herbicide are selected and regenerated into whole organisms.
3. Metabolic/Auxotrophic Marker Genes:
Metabolic or auxotrophic marker genes enable transformed cells to synthesize an essential component, usually an amino acid, which the cells cannot produce otherwise. The surrounding medium is made to intentionally lack the essential component, which cells require to grow.
Cells that have successfully incorporated the selectable marker and the rest of the gene construct will produce the essential components within the cells, and thereby survive. These cells are selected and regenerated into whole organisms.
Screenable Marker Genes:
Screenable markers, also known as assayable markers, are genes which encode for a protein that can be identified through various laboratory assessments. The presence of the protein confirms that transformation has taken place.
Screenable markers are not commonly used because their use is very time-consuming, expensive equipment and other accessories are required to perform the assessments, and the final tissue product has to be destroyed to determine if transformation has taken place.
(c) Multiple Cloning Sites (MCS) or Poly-Linker:
A vector should have a site specific for cloning the foreign DNA fragment provided with one restriction site for most of the commonly used unique restriction endonucleases. All these unique restriction sites
are grouped together in a small region of the vector known as the multiple cloning site (MCS) or the poly-linker.
The presence of unique restriction sites at the MCS gives flexibility in the choice of restriction enzymes.