PLASMIDS

Dr.T.V.Rao MD
 Joshua Lederberg
The term plasmid was first introduced by the American molecular biologist
Joshua Lederberg in 1952

• Joshua Lederberg was an American molecular

biologist known for his work in genetics,
artificial intelligence, and space exploration.
He was just 33 years old when he won the
1958 Nobel Prize in Physiology or Medicine
for discovering that bacteria can mate and
exchange genes. He shared the prize with
Edward L. Tatum and George Beadle who won
for their work with genetics.
Joshua Lederberg
Wins Nobel Prize
 What is a Plasmid
• A plasmid is a DNA molecule that is separate
from, and can replicate independently of, the
chromosomal DNA. They are double stranded
and, in many cases, circular. Plasmids usually
occur naturally in bacteria, but are sometimes
found in eukaryotic organisms (e.g., the 2-
micrometre-ring in Saccharomyces cerevisiae).
 Basic understanding of Plasmid
• A plasmid is a vehicle that can carry artificially
inserted DNA. It will replicate in E. coli, and with its
own replication it will also replicate the inserted
DNA, independent of it's origin. In a way one can see
a plasmid as a minute DNA factory. The main criteria
for a 'good' plasmid is that it takes up the insert you
want to put in, and that it replicates in sufficient
amounts,and that it does not destroy your insert
during the process.
 What is a plasmid?
• A circular piece of
autonomously replicating
DNA

• Originally evolved by
bacteria

• May express antibiotic

resistance gene
or be modified to express
proteins of interest
Bacterial plasmid
 Plasmids

• Plasmids are molecules

of DNA that are found in
bacteria separate from
the bacterial
chromosome.
• They: are small (a few
thousand base pairs)
usually carry only one or
a few genes are circular
have a single origin of
replication
 Multiplication of Plasmids
• Plasmids are replicated by
the same machinery that
replicates the bacterial
chromosome. Some
plasmids are copied at
about the same rate as the
chromosome, so a single
cell is apt to have only a
single copy of the plasmid.
Other plasmids are copied
at a high rate and a single
cell may have 50 or more of
them.
Plasmids enters the Bacteria
with ease.
• Plasmids enter the
bacterial cell with relative
ease. This occurs in nature
and may account for the
rapid spread of antibiotic
resistance in hospitals and
elsewhere. Plasmids can be
deliberately introduced
into bacteria in the
laboratory transforming the
cell with the incoming
genes.
 INTRODUCTION
• Plasmids are self replicative extrachromosomal DNA
molecules of finite size that are stably inherited and
exchanged promiscuously between a broad spectrum of
bacteria and other domains (Perlin, 2002).

• Plasmids allow bacterial (including other domains)

populations to ‘sample’ the horizontal gene pool for
adaptive traits that might be advantageous for survival
under local selective pressure (Sorensen et al., 2005).

• Plasmids also provide genetic variation, acts as sources of

recombination and can allow faster gene fixation leading to
greater likelihood that the ‘new’ trait will persist (Sorensen
et al., 2005).
 INTRODUCTION
• Plasmid classification is generally based on incompatibility group
(determined by their replication/partitioning functions) or the genetic
information specified by their DNA (Perlin, 2002). Incompatibility
grouping had been used to group plasmid of Pseudomonas species
(Jacoby, 1977) and the Enterobacteriaceae into 26 incompatibility group
(Couturier et al., 1988).

• Most plasmids have a narrow host range allowing only intra-species

transfer and replication. However, a small group of plasmids called the
broad host range (BHR) plasmids (Inc P, Q, W, N and C) can be
transferred and replicated in a wide range of bacteria (Hill and Top, 1998;
Dale and Park, 2004). BHR plasmids may either be self-transmissible
(Tra+, Mob+) or mobilizable but not self-transmissible (Tra-, Mob+)
(Perlin , 2002).

• pr
Plasmids are Complex
• Prokaryotes were initially
thought to harbour only circular
plasmids. However with the
discovery of double stranded
linear plasmids in the spirochaete
that cause lyme disease, Borrelia
borgdorferi (Barbour and Garon,
1987), linear plasmids have also
been detected in the genera
Streptomyces (Kinashi et al.,
1994), Mycobacterium (Le
Dantec et al., 2001),
Rhodococcus (Larkin et al.,
2005) and Arthrobacter
(Overhage et al., 2005).
 Understanding a Plasmid
• Many bacteria have accessory
DNA molecules in addition to
their larger chromosome. These
molecules, called plasmids, are
extensively used in genetic
engineering. In order to be
useful in labs, these plasmids
need to have an origin of
replication (ori), which enables
them to replicate within a
bacterial cell. They also need to
have multiple restriction enzyme
sites to enable cutting and
pasting of DNA into a plasmid.
Role in Antibiotic Resistance
• Most plasmids have one
or two identifiable
markers that give a
distinct phenotype to the
bacterial cell. Examples
of such markers include
antibiotic resistance
(ampR) or expression of
an enzyme that catalyzes
a reaction that produces
a color change (lacZ).
 Plasmids and Ampicillin
Resistance
• Some plasmids have the ampR gene,
which confers resistance to the
antibiotic ampicillin. E. coli cells
containing this plasmid, termed
"+ampR" cells, can survive and form
colonies on LB agar that has been
supplemented with ampicillin. In
contrast, cells lacking the ampR
plasmid, termed "–ampR" cells, are
sensitive to the antibiotic, which kills
them. An ampicillin-sensitive cell (–
ampR) can be transformed to an
ampicillin-resistant (+ampR) cell by
its uptake of a foreign plasmid
containing the ampR gene
 Plasmids and Microbes
• In microbiology, an extra chromosomal genetic element that occurs
in many bacterial strains. Plasmids are circular deoxyribonucleic acid
(DNA) molecules that replicate independently of the bacterial
chromosome. They are not essential for the bacterium but may
confer a selective advantage. One class of plasmids, colicinogenic
(or Col ) factors, determines the production of proteins called
colicins, which have antibiotic activity and can kill other bacteria.
Another class of plasmids, R factors, confers upon bacteria
resistance to antibiotics. Some Col factors and R factors can transfer
themselves from one cell to another and thus are capable of
spreading rapidly through a bacterial population. A plasmid that is
attached to the cell membrane or integrated into the bacterial
chromosome is called an episome.
Plasmids in Genetic
Engineering
• Plasmids are extremely
valuable tools in the fields of
molecular biology and
genetics, specifically in the
area of genetic engineering.
They play a critical role in
such procedures as gene
cloning, recombinant protein
production (e.g., of human
insulin), and gene therapy
research. In such procedures,
a plasmid is cut at a specific
site (or sites) using enzymes
called restriction
endonucleases
 Structure of Plasmids
• Plasmid size varies from 1
to over 1,000 kilo base
pairs (kbp). The number of
identical plasmids within a
single cell can range
anywhere from one to even
thousands under some
circumstances. Plasmids can
be considered to be part of the
mobilome, since they are often
associated with conjugation, a
mechanism of horizontal gene
transfer
Plasmids used as Vectors
• Plasmids
• small (1-1000 kb)
• circular
• extrachromosomal DNA

• Growth is independent of
the host’s cell cycle;
amplification of gene
product
• A type of cloning vector
used to carry a gene not
found in the bacterial
host’s chromosome
VARIETIES OF PLASMIDS BASED
ON STRUCTURE
Genetic map of ColE1
circular plasmid: colE1,
imm: genes for production
of, and immunity to colicin
E1; mob codes for nuclease
required for mobilization;
rom codes for protein
required for effective
control of copy number;
oriT: origin of conjugal
transfer; oriV: origin of
replication (Dale and Park,
2004).
 Plasmids can …
• Plasmids are considered transferable genetic
elements, or "replicons", capable of autonomous
replication within a suitable host. Plasmids can be
found in all three major domains, Archea, Bacteria
and Eukarya. Similar to viruses, plasmids are not
considered a form of "life" as it is currently defined.
Unlike viruses, plasmids are "naked" DNA and do not
encode genes necessary to encase the genetic
material for transfer to a new host, though some
classes of plasmids encode the sex pilus necessary
for their own transfer
 Plasmids act as Vectors
• Plasmids are key vectors of
horizontal gene transfer
and essential genetic
engineering tools. They
code for genes involved in
many aspects of microbial
biology, including
detoxication, virulence,
ecological interactions, and
antibiotic resistance.
Plasmids originated several Drug
resistance Mechanisms
• The role of plasmids in evolution of bacterial genome and adaptation to
specific environmental changes has contributed immensely to the
emergence of antibiotic and heavy metal resistance plasmids.

• This feat is aided by the activities of transposons, which promote the

movement of resistance genes between plasmids or from the chromosome
of naturally resistant organisms onto a plasmid ( Dale and Park, 2004;
Kapil, 2005).

• Bacteria can be resistant to various antibiotics either by acquisition of

several independent plasmids or through acquiring a single plasmid with
many resistance determinant on it (Hill and Top, 1998).
 VARIETIES OF PLASMIDS BASED ON FUNCTIONS

Pumping of the toxic metal out of the bacterial cell,

bioaccumulation in physiologically inaccessible compound
and redox chemistry in which a more toxic ion species is
converted to a less toxic ion (Endo et al., 2002)

• Plasmid-mediated mercury (mer) (Silver and Walderhaug,

1994), arsenic (ars) (Wu and Rosen, 1993), cadmium
(Cad) (Tsai et al., 1993), and chromate (Chr) (Pimentel et
al., 2002) resistance have been reported for Gram positive
and Gram negative with various mechanisms of resistance.
VARIETIES OF PLASMIDS
BASED ON FUNCTIONS
• Genetic map of Escherichia coli
F factor showing the four major
regions: The inc, rep region
determines replication and
plasmid incompatibility
properties; the tra region
provides conjugative DNA
mobilization functions; the
region containing the four
transposable elements that
facilitates interaction between F
factor and other DNA molecules;
and the silent region (Porter,
2002).
Escherichia coli Cloning and
Expression Vectors
• Gene technology methods
together with the DNA
sequencing and PCR
methodologies have
revoluzionarized basic and
applied research in molecular
biology involving all kinds of
organisms from eubacteria up to
humans. Since almost all of
the cloning experiments
start by insertion of DNA
fragments into Escherichia
coli plasmid
VARIETIES OF PLASMIDS BASED ON FUNCTIONS
Table 1: some antibiotic resistance plasmids, their
incompatibility groups and phenotypes

Abbreviations used: Km, kanamycin; Tc, tetracycline; Ap, ampicillin; Sm,

streptomycin; Sp, spectinomycin; Cm, chloramphenicol, Tp, trimethoprim; BHR,
broad host range (Hill and Top, 1998)
 VARIETIES OF PLASMIDS BASED ON FUNCTIONS
Sex pheromone plasmids

• Pheromones are secreted chemicals used for signalling between two or

more individuals. Pheromone conjugative plasmids are confined to the
Enterococci and encode antibiotic resistance, bacteriocins and hemolysins
(Grohmann et al., 2003).

• In this novel transfer system, recipient cells secrete a family of heat stable
peptide pheromones with specificities for donor carrying various
conjugative plasmid that trigger response from donor bacteria harbouring a
particular plasmid, which synthesize an adhesin that facilitates the
formation of mating aggregate with nearby recipients (Dale and Park,
2004).

• Examples of sex pheromone plasmids are pAD1 (59.3-kb,

hemolysin/bacteriocin plasmid), and pCF10 (65-kb, tetracycline resistance)
(Grohmann et al., 2003).
 VARIETIES OF PLASMIDS BASED ON FUNCTIONS
Senescence plasmids in fungi
• Senescence is an inherent degenerative program in multicellular organisms
that is manifested by a progressive decline in cellular energy production
culminating in the death of a part or the whole organism (D’souza and
Maheshwari, 2002).

• This phenomenon was found to be associated with the accumulation of high

copy number circular plasmids (sen DNAs) in the mitochondrial respiration
resulting in the death of the fungus (D’souza and Maheshwari, 2002).

• Mitochondrial-based linear plasmids from Neurospora sp. were also found to

be responsible for the death of the strains harbouring them by insertion of
their DNA into the mitochondrial genome resulting in disruption of several
genes leading to senescence and death due to defective respiration (Bertrand,
2000; Griffiths, 1998).
1973 Genetic Engineering Invented
 Cloning: Digestion

TARGET GENE

SOURCE DNA
Cloning: Digested Fragments

TARGET GENE
Cloning: Ligation
DNA LIGASE

TARGET GENE
Cloning: Recombinant
Plasmid
TARGET GENE
Cloning: Transformation

E. coli
Cloning: Competence
Ca2+ Ca2+ Ca2+ Ca2+
Ca2+
Ca2+ Ca2+ Ca2+ Ca2+
Ca2+ Ca2+ Ca2+
Ca2+
Ca2+ Ca2+
Ca2+
Ca2+
E. coli
Ca2+ Ca2+
Ca2+
Ca2+
Ca2+
Ca2+ Ca2+ Ca2+ 2+ Ca2+ Ca2+
Ca Ca2+
Ca2+
Cloning: Competence

E. coli
Cloning: Transformation

E. coli
Cloning: Transformation

E. coli
Plasmids in Antibiotic Resistance
• In addition plasmids carry
antibiotic resistance genes
and their spread in
pathogenic bacteria is of
great medical importance.
Plasmids are used in
molecular studies of
various organisms with
ramifications in synthetic
biology, medicine, ecology
Antibiotic resistance
• Plasmid often contain genes
or gene-cassettes that confer
a selective advantage to the
bacterium harboring them,
e.g., the ability to build an
antibiotic resistance. Every
plasmid contains at least one
DNA sequence that serves as
an origin of replication or ori
(a starting point for DNA
replication), which enables
the plasmid DNA to be
duplicated independently
from the chromosomal DNA
 Plasmids can be Engineered for
Selective Needs
• While there are only a couple essentials that a
plasmid must have, DNA workers have engineered a
number of features into many plasmids that make
them easier to use. First the essentials: 1) it must
have the _ori_ gene, which enables the plasmid to be
replicated in the host cells, 2) it must have a
"selectable marker" so that only cells that have the
plasmid will grow in culture. This is almost always a
gene that confers resistance to a particular antibiotic,
so that cells grown in the presence of the drug will all
contain the plasmid.
 Plasmid-based Expression
Systems for Mammalian Cells
• In contrast to bacteria and yeast
no natural plasmids are found in
mammalian cells. Therefore
many attempts to construct
different expression vector
systems for mammalian cells
have been made in recent years.
These vector systems can be
categorized in terms of vector
administration, mechanisms of
vector replication and
mechanisms to achieve nuclear
persistence of the vectors
 Limitations of Plasmids
• There are limitations to each of these steps: large
inserts require specialized plasmids (cosmids or YACs
for megabase sized- inserts), the larger a
plasmid+insert, the lower its replication rate, but
there are ways to improve the yield, and certain
plasmids result in frequent deletions of (parts of) the
insert, although this is sometimes due to the host
(the E. coli or another host cell) or due to the nature
of the insert as well.
 Plasmids in Gene therapy
• The success of gene therapy depends on the efficient
insertion of therapeutic genes at the appropriate
chromosomal target sites within the human genome,
without causing cell injury, oncogenic mutations or
an immune response. Although viral vectors offer
excellent vehicles for highly efficient transduction of
human cells, the associated safety concerns make
non-viral delivery of therapeutic genes by using
plasmid DNA into cells more attractive.
Dual Expression Cassette Plasmids for the Expression
of One Gene of Interest pSELECT
• pSELECT plasmids offer all the
features necessary to express a
gene of interest at high levels in
a large number of cell types.
pSELECT plasmids contain two
transcription units, the first
drives the expression of the gene
of interest and the second drives
the expression of a large choice
of dominant selectable markers
for both E. coli and mammalian
cells.
 Horizontal Gene Transfer
Mediated by Plasmids
• Among the mobile elements and mechanisms of HGT,
plasmids are undoubtedly critical players because of
their ability to transfer by conjugation among both
closely and very distantly related bacterial hosts. This
feature allows them to broadly distribute genes or
gene clusters that code for various host-beneficial
phenotypes. The event, generally recognized as
horizontal gene transfer (HGT), is now considered as
a strong driving force for the evolution of bacterial
genome organization and for rapid adaptation to the
surrounding environments
 Plasmid DNA as Prophylactic and Therapeutic
vaccines for Cancer and Infectious Diseases
• Concept of plasmid DNA
as a protective vaccine
strategy for cancer and
infectious diseases.
However, the crossover
application into human
studies has been met
with poor results based
on the DNA vector¹s
inability to provide
clinically relevant
prophylactic and
therapeutic benefit
 Plasmid profiles in epidemiologic surveillance of
disease outbreaks and Drug Resistance

• Plasmids can also serve as

markers of various bacterial
strains when a typing system
referred to as plasmid profiling,
or plasmid fingerprinting is
used. Because many species of
bacteria contain plasmids,
plasmid profile typing has been
used to investigate outbreaks of
many bacterial diseases and to
trace inter- and intra-species
spread of antibiotic resistance.
Programme created by Dr.T.V.Rao
MD for ‘e’ learning for Medical
and paramedical students in
Developing World
Email
doctortvrao@gmail.com