Cloning Vectors and pBR322 | Biotechnology: Principles and Processes | Class 12 Biology | NEET

Plasmids and bacteriophages are used as cloning vectors.
This is because plasmids and bacteriophages have the
ability to replicate within the bacterial cell independent of
chromosomal DNA.
Bacteriophages have very high copy numbers of their
genome within the bacterial cells. But in case of plasmids,
some may have only one or two copies per cell whereas
others may have 15-100 copies per cell.
Cloning vectors use the machinery of bacterial cell to
replicate and thereby, increase the copy number (make
clones) of the DNA inserted into them.
These help easy linking of foreign DNA and allow the
selection of recombinants (bacterial cells that have picked
up recombinant plasmid) from non-recombinants (those
who have not).
All vectros have four special features that are required to
facilitate cloning into a vector.
Origin of replication (ori) is the sequence from where
replication starts and any piece of DNA when linked to
this sequence can be made to replicate within the host
cells. This sequence also controls the copy number of the
linked DNA.
Selectable marker helps in identifying and eliminating
non-transformants and selectively permitting the growth
of the transformants.
Transformation is the procedure through which a piece
of DNA is introduced into a host bacterium.
Normally, antibiotics such as ampicillin,
chloramphenicol, tetracycline or kanamycin, etc., are
considered useful selectable markers.
Cloning sites are used to link the alien DNA. The vector
needs to have cloning or recognition sites for the
commonly used restriction enzymes. The presence of more
than one recognition sites within the vector will generate
several fragments and complicate the gene cloning. The ligation of foreign DNA is carried out at a restriction
site present in one of the two antibiotic resistance
genes. It can be done at the Bam HI site of tetracycline
resistance gene in the vector pBR 322.
In this case, the recombinant plasmids will lose
tetracycline resistance due to the insertion of foreign
DNA but can still be selected out from non-recombinant
ones by plating the transformants on a medium
containing ampicillin.
The recombinants will grow in ampicillin containing
medium but not on that containing tetracycline. But, non-recombinants will grow on the medium containing
both the antibiotics.
Other selectable markers can differentiate recombinants
from non-recombinants on the basis of their ability to
produce colour in the presence of a chromogenic
substrate. In this, recombinant DNA is inserted within
the coding sequence of an enzyme, β-galactosidase.
This results into inactivation of the gene synthesis of this
enzyme and referred to as insertional inactivation.
The presence of a chromogenic substrate gives blue
coloured colonies if the plasmid in the bacteria does not
have an insert and if the colonies have inserted plasmid,
such colonies are recombinants and show no
colouration.
Vectors for cloning genes in plants and animals A
pathogen of several dicot plant is able to deliver a piece of
DNA, i.e. ‘T-DNA’ to transform normal plant cells into a
tumour cells and disect these tumour cells to produce the
chemicals required by the pathogen. Similarly, retrovirus in
animals have the ability to transform normal cells into
cancerous cells.
The tumour inducing (Ti) plasmid of Agrobacterium
tumefaciens has now been modified into a cloning vector
which is able to use the mechanisms to deliver genes of our
interest into a variety of plants.
Similarly, retroviruses can be disarmed and used to deliver
desirable genes into animal cells.

#neet #biology #cuet #class12