Plasmid
Plasmids are (typically) circular double-stranded DNA molecules that are
separate from the chromosomal DNA (Fig. 1). They usually occur in
bacteria, sometimes in eukaryotic organisms (e.g., the 2-micrometre-ring in
Saccharomyces cerevisiae). Their size varies from 1 to over 400 kilobase
pairs (kbp). There are anywhere from one copy, for large plasmids, to
hundreds of copies of the same plasmid present in a single cell.
Figure 1: Schematic drawing of a bacterium with plasmids enclosed.
(1)Chromosomal DNA. (2) Plasmids
What is a Bacterial Plasmids?
A plasmid is an extra-chromosomal element, often a circular
DNA. The plasmids typically have three important elements:
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Coiling in a
plasmid
An origin of replication
A selectable marker gene (e.g. resistance to
ampicillin)
A cloning site (a place to insert foreign DNAs)
The plasmids are double-stranded DNA .DNA has the
double-helical structure that, DNA can take on a higher order
coiling that twists one double helix around another. We call
this "superhelical coiling" or simply "supercoiling." In a
linear molecule these twists can unravel by themselves,
provided the ends are not prevented from rotating. In a
circular molecule with no free ends, the superhelical twists
are "locked in" and the molecule cannot relax. This coiling is
not the same as the right-handed double helix coil with
which you are all familiar. The supercoiled molecule is a
coiled coil.
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A supercoiled circular DNA.
Supercoiled DNA
Relaxation
What's needed to get supercoiled circular DNA to relax? If
one of the two strands is broken so that it has free 5' and 3'
ends, the supercoils can relax even though the overall
structure of the molecule remains a circle. The free ends of
the broken strand rotate around the phosphate backbone of
the intact strand (the one that wasn't broken). This loss of
superhelical stress puts the plasmid into a "relaxed DNA"
form.
Another electron micrograph: This one is of relaxed DNA
Relaxed DNA
a "supercoil", is illustrated at the left:
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Episomes
Episomes are plasmids that can integrate themselves into the chromosomal
DNA of the host organism (Fig. 3). For this reason, they can stay intact for a
long time, be duplicated with every cell division of the host, and become a
basic part of its genetic makeup. This term is no longer commonly used for
plasmids, since it is now clear that a region of homology with the
chromosome such as a transposon makes a plasmid into an episome.
While some plasmids like to insert themselves into the chromosome as
"episomes" (what's an epi-phyte or an epi-dermis?), these still have a phase
in which they are by themselves in the cytoplasm - and they are circular
double=helices.
How these circular helices get from cell to cell depends on whether they are
viral or plasmid. If they are viral they code for extracellular packaging layers
so that they can float through the environment and attach to another
appropriate cell.
If it is plasmid DNA, then there are genes coding for cellular equipment that
can be used in sort of a sexual way to duplicate the plasmid DNA and then
send one copy through a tube into a cell that doesn't have that particular type
of plasmid. Obviously, the plasmid DNA must also code for some surface
components of the host cell so that other donors of this plasmid don't try to
"mate" with this cell.
While most viruses make their cells "sick" or even kill them, plasmids rarely
do harm to their host cells. Usually they multiply up to some small number
(called the "copy number") and stop reproducing. They usually confer some
sort of benefit to their hosts - new metabolic capacities, or changing the
surfaces of the hosts so that fewer types of viruses can infect them.
Vectors
Plasmids used in genetic engineering are called vectors. They are used to
transfer genes from one organism to another and typically contain a genetic
marker conferring a phenotype that can be selected for or against. Most also
contain a polylinker or multiple cloning site (MCS), which is a short region
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containing several commonly used restriction sites allowing the easy
insertion of DNA fragments at this location.
Figure 3: Comparison of non-integrating plasmids (top) and episomes
(bottom). 1 Chromosomal DNA. 2 Plasmids. 3 Cell division. 4
Chromosomal DNA with integrated plasmids
Plasmids
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are small - a few thousand base pairs(about 2,000 to 10,000 base
pairs)
usually carry only one or a few genes
are circular
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
have a single origin of replication
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.
Genes on plasmids with high numbers of copies are usually expressed at
high levels. In nature, these genes often encode proteins (e.g., enzymes) that
protect the bacterium from one or more antibiotics.
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.
A plasmid is a small circular piece of DNA (about 2,000 to 10,000 base
pairs) that contains important genetic information for the growth of bacteria.
In nature, this information is often a gene that encodes a protein that will
make the bacteria resistant to an antibiotic. Plasmids probably came about as
a result of bacteria evolving in close proximity to other heterotrophs.
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Bacteria often grow in the same environment as molds and fungi and
compete with them for food (complex organic material). As a result, molds
and fungi have evolved to make toxins that kill bacteria (which we now use
as antibiotics in medicine) in order to win in the competition for food.
Bacteria, in turn, evolved to make proteins that inactivate the toxins. The
bacteria share this vital information by passing it among themselves in the
form of genes in plasmids.
Plasmids were discovered in the late sixties, and it was quickly realized
that they could be used to amplify a gene of interest. A plasmid containing
resistance to an antibiotic (usually ampicillin) is used as a vector. The gene
of interest is inserted into the vector plasmid and this newly constructed
plasmid is then put into E. coli that are sensitive to ampicillin. The bacteria
are then spread over a plate that contains ampicillin. The ampicillin provides
a selective pressure because only bacteria that have acquired the plasmid can
grow on the plate. Therefore, as long as you grow the bacteria in ampicillin,
it will need the plasmid to survive and it will continually replicate it, along
with your gene of interest that has been inserted to the plasmid.
There are many different kinds of plasmids commercially available. All of
them contain 1) a selectable marker (i.e., a gene that encodes for antibiotic
resistance), 2) an origin of replication (which is used by the DNA making
machinery in the bacteria as the starting point to make a copy of the
plasmid) and 3) a multiple cloning site. The multiple cloning site has many
restriction enzyme sites and is used to insert the DNA of interest. The
multiple cloning site is usually in the middle of a reporter gene like Lac Z. A
commonly used plasmid is pBluescript:
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Figure 1
The main differences among commercially available plasmids are the
number of restriction enzyme sites, their order in the multiple cloning site,
the type of antibiotic resistance that the plasmid confers, and some other
genetic information that makes the plasmid useful for a specific purpose.
Bacteria transformed with pBluescript will survive in ampicillin containing
media and will replicate the plasmid, including any gene that is placed in the
multiple cloning site.
Competent Cells:
Since DNA is a very hydrophilic molecule, it won't normally pass through a
bacterial cell's membrane. In order to make bacteria take in the plasmid, they
must first be made "competent" to take up DNA. This is done by creating
small holes in the bacterial cells by suspending them in a solution with a
high concentration of calcium. DNA can then be forced into the cells by
incubating the cells and the DNA together on ice, placing them briefly at
42oC (heat shock), and then putting them back on ice. This causes the
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bacteria to take in the DNA. The cells are then plated out on antibiotic
containing media.
Why Plasmids are Good Cloning Vectors
small size (easy to manipulate and isolate)
circular (more stable)
replication independent of host cell
several copies may be present (facilitates replication)
frequently have antibody resistance (detection easy)
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Antibiotic resistance genes are carried on
plasmids
In 1968 when I arrived on the Stanford faculty
as an Assistant Professor, I set out to isolate
and characterize the plasmids of infectious
drug resistance with the hope of answering
these questions. Work carried out in my
laboratory and elsewhere soon showed that
antibiotic resistance plasmids were molecules
of circular DNA.
Breaking open bacterial cells released these DNA circles as seen in this
electron photo micrograph taken some years ago at Stanford by Dr. Jack
Griffith. These circles could then be isolated and characterized by
biochemical methods. Studies showed that plasmids contain a DNA
segment called the replication region which allows the plasmid to
propagate itself independently of the machinery that reproduces the
chromosomal DNA.
Plasmid Ancillary Genes include:
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Antibiotic-resistance genes
Antibiotics production genes
Heavy Metal resistance genes
Virulence genes
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Tumorigenicity (in plants)
Fertility (transfer) genes
Toxin production
Restriction / Modification
Metabolism of hydrocarbons
In different plasmids the replications regions are linked to different
ancillary genes, encoding traits that ordinarily are not essential to the
bacterial host but which can, in some environments, provide a biological
advantage to bacteria carrying the plasmid. Antibiotic resistance is one of
these traits. In some plasmids the replication region is attached to genes
that produce antibiotics or toxins. In others, the genes that enable to
plasmid to be transferred between bacteria are other genes the plasmid has
picked up from host cells.
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University of Kentucky
Plasmid Vector Catalog
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pGEM-3Zf+: Promega
This vector can be used as a standard cloning vector, as
templates for in vitro transcription, and for production of
circular ssDNA for sequencing due to the presence of the
origin of replication of the filamentous phage f1. The
presence of the gene encoding the lacZ a-peptide allows
recombinants to be selected by blue-white screening.
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pT7blue: Novagen
pT7blue contains the pUC19 backbone, a T7 promoter, f1
origin of replication, and modified multiple cloning
region. The multiple cloning region contains an EcoRV
site used for blunt cloning flanked by an NdeI site, which
allows PCR fragments to be conveniently subloned into
the NdeI sites of many pET vectors.
pUC19 : pUC19 is a small, high copy number E.coli plasmid
cloning vector that is part of a series of related plasmids
constructed by Messing and co-workers (Yanisch-Perron et al.,
Gene 33, 103-119). The pUC plasmids contain portions of
pBR322 and M13mp19.
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pZL1: Life Technologies
pZL1 is an E. coli cloning vector derived from pSPORT 1
containing a loxP sequence installed at one of the BspHI
sites and the phage P1 incA incompatibility locus at the
other BspHI site. All other features of pSPORT are
ZIPLOXTM DNA by cre-mediated recombination.
No picture available.
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pBR322
pBR322 carries genes that confer tetracycline and
ampicillin resistance. It was constructed from several
naturally occuring plasmids (Balbas et al., Gene 50: 3).
No picture available.
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pBluescript II KS+: Stratagene
The pBluescript phagemid was derived from pUC19. The
KS designation indicates that the polylinker is oriented so
that lacZ transcription proceeds from KpnI to SacI. The
phagemid contains an f1 origin of replication, a ColE1
origin, the lacZ gene interupted by the polylinker region
to facilitate blue-white screening, and an ampicillin
resistance gene.
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JUMPING GENES! (Transposons)
Evolutionary Leaps - Part Two
We have seen how viruses and plasmids can move genes around within the
branches of the Shrub of Life. But let us suppose that somewhere along this
convoluted pathway by which some block of genes are moving around, it
comes to a cell that contains a transposon. That transposon often can pick up
a neighboring host gene and jump with it into either a plasmid or a virus
genome. And that plasmid or virus containing the transposon then is moved
to a new closely related cell type, where the transposon and its associated
gene, jump off (damaging the viral or plasmid DNA, but nevertheless getting
inserted into the new host's chromosome - conferring it with a new gene).
Whether that gene or block of genes is used immediately or not, determines
the rate of this step of evolution. Most of us carry large amounts of unused
DNA - just waiting for some future eon to be found useful and cause a leap
in evolution.
c. Plasmids and Transposons
The overall purpose of this Learning Object is:
1) to learn the chemical makeup and the functions associated with of
bacterial plasmids; and
2) to introduce the relationship between bacterial plasmids and the transfer
of antibiotic resistance from one bacterium to another.
Plasmids (def) and Transposons (def)
In addition to the nucleoid, many bacteria often contain small
nonchromosomal DNA molecules called plasmids. Plasmids usually contain
between 5 and 100 genes. Plasmids are not essential for normal bacterial
growth and bacteria may lose or gain them without harm. They can,
however, provide an advantage under certain environmental conditions.
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For example, under normal environmental growth conditions, bacteria are
not usually exposed to antibiotics and having a plasmid coding for an
enzyme capable of denaturing a particular antibiotic is of no value.
However, if that bacterium finds itself in the body when the particular
antibiotic that the plasmid-coded enzyme is able to degrade is being given to
treat an infection, the bacterium containing the plasmid is able to survive and
grow.
composition: Plasmids are small molecules of double stranded, helical,
nonchromosomal DNA. Like the nucleoid, the two ends of the doublestranded DNA molecule that make up a plasmid covalently bond together
forming a physical circle.
function: Plasmids code for synthesis of a few proteins not coded for by
the nucleoid. For example, R-plasmids, found in some gram-negative
bacteria, often have genes coding for both production of a conjugation pilus
(discussed later in this unit) and multiple antibiotic resistance. Through a
process called conjugation, the conjugation pilus enables the bacterium to
transfer a copy of the R-plasmids to other bacteria, making them also
multiple antibiotic resistant and able to produce a conjugation pilus. In
addition, some exotoxins , such as the tetanus exotoxin and Escherichia coli
enterotoxin discussed later in this unit under Bacterial Pathogenicity, are
also coded for by plasmids.
For a detailed look at plasmids, see the online textbook
Microbiology Webbed Out at the University of WisconsinMadison.
Transposons (transposable elements or "jumping genes") are small pieces of
DNA that encode enzymes that transpose the transposon, that is, move it
from one DNA location to another. Transposons may be found as part of a
bacterium's nucleoid (conjugative transposons) or in plasmids and are
usually between one and twelve genes long. A transposon contains a number
of genes, coding for antibiotic resistance or other traits, flanked at both ends
by insertion sequences coding for an enzyme called transpoase. Transpoase
is the enzyme that catalyzes the cutting and resealing of the DNA during
transposition. Thus, such transposons are able to cut themselves out of a
bacterial nucleoid or a plasmid and insert themselves into another nucleoid
or plasmid and contribute in the transmission of antibiotic resistance among
a population of bacteria.
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Plasmids can also acquire a number of different antibiotic resistance genes
by means of integrons. Integrons are transposons that can carry multiple
gene clusters called gene cassettes that move as a unit from one piece of
DNA to another. An enzyme called integrase enables these gene cassettes
to integrate and accumulate within the integron. In this way, a number of
different antibiotic resistance genes can be transferred as a unit from one
bacterium to another.
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Plasmids
A
plasmid
is
an
independent,
circular,
self-replicating
DNA
molecule that carries only
a few genes. The number
of plasmids in a cell
generally remains constant from generation to
generation. Plasmids are autonomous molecules and
exist in cells as extrachromosomal genomes, although
some plasmids can be inserted into a bacterial
chromosome, where they become a permanent part of
the bacterial genome. It is here that they provide great
functionality in molecular science.
Plasmids are easy to manipulate and isolate using
bacteria (see also alkaline lysis) They can be integrated
into mammalian genomes, thereby conferring to
mammalian cells whatever genetic functionality they
carry. Thus, this gives you the ability to introduce genes
into a given organism by using bacteria to amplify the
hybrid genes that are created in vitro. This tiny but
mighty plasmid molecule is the basis of recombinant
DNA technology.
There are two categories of plasmids. Stringent
plasmids replicate only when the chromosome
replicates. This is good if you are working with a protein
that is lethal to the cell. Relaxed plasmids replicate on
their own. This gives you a higher ratio of plasmids to
chromosome.
So how do we manipulate these plasmids?
1. Mutate them using restriction enzymes, ligation
enzymes, and PCR. Mutagenesis is easily
accomplished by using restriction enzymes to cut
out portions of one genome and insert them into a
plasmid. PCR can also be used to facilitate
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mutagenesis. Plasmids are mapped out indicating
the locations of their origins of replication and
restriction enzyme sites.
2. Select them using genetic markers. Some
bacteria are antibiotic resistant. While this is a
serious health problem, it is a godsend to
molecular scientists. The gene that confers
antibiotic resistance can be added (ligated) to the
gene you are inserting into the plasmid. So every
plasmid that contains your target gene will not be
killed by antibiotics. After you transfect your
bacterial cells with your engineered plasmid (the
one with the target gene and the antibiotic resistant
marker), you incubate them in a nutrient broth that
also contains antibiotic (usually ampecillin). Any
cells that were not transfected (this means they do
not have your target gene in them) are killed by
the antibiotic. The ones that do have the gene also
have the antibiotic resistant gene, and therefore
survive the selection process.
3. Isolate them (such as with alkaline lysis)
4. Transform them into cells where they become
vectors to transport foreign genes into a recipient
organism.
There are some minimum requirements for plasmids that
are useful for recombination techniques:
1. Origin of replication (ORI). They must be
able to replicate themselves or they are of no
practical use as a vector.
2. Selectable marker. They must have a marker
so you can select for cells that have your plasmids.
3. Restriction enzyme sites in non-essential
regions. You don't want to be cutting your
plasmid in necessary regions such as the ORI.
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In addition to these necessary requirements, there are
some factors that make plasmids either more useful or
easier to work with.
1. Small. If they are small, they are easier to
isolate (you get more), handle (less shearing), and
transform.
2. Multiple restriction enzyme sites. More sites
give you greater flexibility in cloning, perhaps
even allowing for directional cloning.
3. Multiple ORIs. It is important to note that two
genes must have different ORIs if they are going
to be inserted in the same plasmid.
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