INSTITUTE FOR

EXELLENCE IN HIGHER
EDUCATION

PLASMID

by Arwa Hussain
BSc 3 rd year
Biotechnology (honours)
 VECTORS
Vector is an agent that can carry a DNA fragment into a host
cell. If it is used for reproducing the DNA fragment, it is
called a “cloning vector”. If it is used for expressing certain
gene in the DNA fragment, it is called an “expression
vector”. A cloning vector is a small piece of DNA into which
a foreign DNA fragment can be inserted. There are many
types of cloning vectors. Plasmids and bacteriophages are
the most commonly used for this purpose.
 PLASMIDS
An extra chromosomal circular double stranded
DNA molecule that autonomously replicates
inside the bacterial cell and the cloning limit is
100 to 10,000 bp. The term “plasmid” was first
introduced by the American molecular
biologist, Joshua Lederberg in 1952.
 Classification of Plasmid
 Fertility or F plasmid: These type of plasmids carry only
tra genes and have no characterstic feature beyond the
ability to promote conjugal transfer of plasmids. A well-
known example is the F plasmid of E.coli.
 Resistance or R plasmids: These carry genes conferring on
the host bacterium resistance to one or more antibacterial
agents, such as chloramphenicol, ampicillin, and mercury. R
plasmids are very important in clinical microbiology as their
spread through natural populations can have profound
consequences in treatment of bacterial infections. An
example is RP4, which is commonly found in
Pseudomonas, but also occurs in many other bacteria.
 Col plasmids: Col plasmids code for colicins, proteins
that kill other bacteria. An example is ColE1 of E. coli.
 Degradative plasmids: These type of plas6mids allow
the host bacterium to metabolize unusual molecules
such as toulene and salicylic acid, an example being
TOL of Pseudomonas putida.
 Virulence plasmids: These plasmid confer
pathogenicity on host bacterium; these include the Ti
plasmids of Agrobacterium tumefaciens, which induce
crown gall disease on dicotyledonous plants.
 Classification on the basis of number of
copies
 Relaxed plasmids: which are normally
maintained at multiple copies per cell.
 Stringent plasmids: which have a limited
number of copies per cell.
 Replication of Plasmid
 The independent replication of plasmid is due to the
presence of certain sequence acting as the origin of
replication.
 The smaller plasmids use the DNA replicative enzymes of
the host cells while the larger plasmids carry genes that
code for special enzymes necessary for their replication.
 Some plasmid can integrate themselves into the bacterial
chromosome. They are called episomes or integrative
plasmids.
 These plasmids replicate along with the bacterial
chromosome.
 Isolation of Plasmid
 Growth of bacterial and plasmid
ampliflication.
 Breaking the bacterial cells to release their
contents.
 Treatment of bacterial cell extracts to remove
all components except the DNA.
 Separation of Plasmid DNA from the
chromosomal DNA.
 Properties of ideal cloning vector
 A plasmid vector should be small.
 The plasmid should have selectable markers
which can be used to distinguish between
transformed or non-transformed cell.
 The plasmid should easily propagate inside the
desired host so that number of recombinant
DNA molecule can be amplified.
 Examples
Plasmid pBR322
pBR 322 is an artificial plasmid, which is regarded as the
‘work house’ of gene cloning laboratory. Its DNA is derived
from different but naturally occurring plasmids.
pBR322 has restriction sites for over 20 restriction
endonucleases and it containes genes that gives resistance
against antibiotic ampicillin and tetracycline. Hence cloning
of pBR322 at any of the sites within these gene will result in
insertional inactivation of either of these gene.
pUC vectors
Another series of plasmids that are used as cloning vectors belongs
to pUC series. These plas6mids are 2700 bp long and possess.
ampicillin resistance gene,
the origin of replication derived from pBR322 and
the lac Z gene derived from E. coli.
When DNA fragment are cloned in this region of pUC, the lac Z
gene is inactivated. These plasmid when transfor6med into E.
coli. Strain, which is lac Z
and grown in presence of IPTG and X- gal will rise to white or
clear colony. On other hand, pUC having no inserts and
transformed into bacteria will have active lac Z gene therefore
will produce blue colonies.