Plasmids

PLASMIDSDr.T.V.Rao MD

Joshua LederbergThe 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 DNAmolecules of finite size that are stably inherited andexchanged promiscuously between a broad spectrum ofbacteria and other domains (Perlin, 2002).

• Plasmids allow bacterial (including other domains)populations to ‘sample’ the horizontal gene pool foradaptive traits that might be advantageous for survivalunder local selective pressure (Sorensen et al., 2005).

• Plasmids also provide genetic variation, acts as sources ofrecombination and can allow faster gene fixation leading togreater likelihood that the ‘new’ trait will persist (Sorensenet 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 therapyresearch. 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 vectorused to carry a gene not found in the bacterial host’s chromosome

VARIETIES OF PLASMIDS BASED

ON STRUCTUREGenetic 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


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)


Sex pheromone plasmids

• Pheromones are secreted chemicals used for signalling between two ormore individuals. Pheromone conjugative plasmids are confined to theEnterococci and encode antibiotic resistance, bacteriocins and hemolysins(Grohmann et al., 2003).

• In this novel transfer system, recipient cells secrete a family of heat stablepeptide pheromones with specificities for donor carrying variousconjugative plasmid that trigger response from donor bacteria harbouring aparticular plasmid, which synthesize an adhesin that facilitates theformation 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).


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

TARGET GENE

DNA LIGASE

Cloning: Recombinant

Plasmid

TARGET GENE

Cloning: Transformation

E. coli

Cloning: Competence

E. coli

Ca2+

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Ca2+

Ca2+

Ca2+ Ca2+Ca2+

Ca2+

Ca2+Ca2+Ca2+

Ca2+

Ca2+

Ca2+Ca2+

Ca2+

Ca2+

Ca2+

Ca2+ Ca2+Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+ Ca2+

Cloning: Competence

E. coli


E. coli


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 genesor 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.

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Plasmids