VECTOR
A Vector is a DNA molecule that has the ability to replicate
autonomously in an appropriate host cell and into which the DNA fragment to be
cloned (called DNA insert) is integrated for cloning. Therefore, a vector must
have an origin of DNA replication (denoted as ori) that functions in the host cell.
Any extra-chromosomal small genome, e.g., plasmid, phage or virus, may be used
as a vector.
Properties of A Good Vector
A good vector must have the following properties.
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. A vector should be ideally less than 10 kb in size
because large DNA molecules are broken during purification procedure. In
addition, large vectors present difficulties during various manipulations
required for gene cloning
3. The vector should be easy to isolate and purify.
4. It should be easily introduced into the host cells, i.e.,
transformation of the host with the vector should be easy.
5.The vector should have suitable marker genes that allow
easy detection and/or selection of the transformed host cells.
6. 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.
7. The cells transformed with such vector molecules that
contain the DNA insert (recombinant DNA) should be identifiable or selectable
from those transformed by the vector molecules only.& A vector should
contain unique target sites for as may restriction enzymes as possible into which
the DNA insert can be integrated without disrupting an essential function.
9. 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; several other features may also be important.
It should be kept in
mind that (
1) the DNA molecules used as vectors have coevolved with
their specific natural host species, and hence are adapted to function well in
them and in their closely related species. Therefore, the choice of vector
depends largely on the host species into which the DNA insert or gene is to be
cloned. In addition,
(2) most naturally occurring
vectors do not have all the required functions; therefore, useful vectors have
been created by joining together segments performing specific functions (called
modules) from two or more natural entities. A brief description of some of the
important vectors used in different hosts is given below.
Cloning And
Expression Vectors
All vectors used for propagation of DNA inserts in a
suitable host are called cloning vectors. But when a vector is designed for the
expression of, i.e., production of the protein specified by, the DNA insert, it
is termed as expression vector. As a rule, such vectors contain at least the
regulatory sequences, i.e., promoters, operators, ribosomal binding sites,
etc., having optimum function in the chosen host. It is desirable that all
cloning vectors have relaxed replication control so that they can produce
multiple copies per host cell.
When an eukaryotic gene is to be expressed in a prokaryote,
the eukaryotic coding sequence has to be placed after prokaryotic promoter and
ribosome binding site, since the regulatory sequences of eukaryotes are not
recognised in prokaryotes. In addition, eukaryotic genes, as a rule, contain
introns (noncoding regions) present within their coding regions. These introns
must be removed to enable the proper expression of eukaryotic genes since
prokaryotes lack the machinery needed for their removal from the RNA
transcripts. When eukaryotic genes are isolated as cDNA, they are intron-free
and suitable for expression in prokaryotes.
Several strategies have been attempted for the construction
of expression vectors using regulatory sequences of the appropriate hosts.
These approaches may be grouped into the following two broad categories.
1. Construction of vectors allowing the synthesis of fusion proteins
comprising amino acids encoded by a sequence in the vector and those encoded by
the DNA insert (translational fusion).
2. Development of vectors permitting the synthesis of pure
proteins encoded exclusively by the DNA inserts (transcriptional fusion).