Download Types of plasmid One way of grouping plasmids is by their ability to

Types of plasmid
One way of grouping plasmids is by their ability to transfer to other
bacteria. Conjugative plasmids contain so-called tra-genes, which perform
the complex process of conjugation, the sexual transfer of plasmids to
another bacterium (Fig. 4). Non-conjugative plasmids are incapable of
initiating conjugation, hence they can only be transferred with the assistance
of conjugative plasmids, by 'accident'. An intermediate class of plasmids are
mobilisable, and carry only a subset of the genes required for transfer. These
plasmids can 'parasitise' another plasmid, transferring at high frequency in
the presence of a conjugative plasmid.
Figure 4 : Schematic drawing of bacterial conjugation. 1 Chromosomal
DNA. 2 Plasmids. 3 Pilus.
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It is possible for several different types of plasmids to coexist in a single
cell, e.g., seven different plasmids have been found in E. coli. On the other
hand, related plasmids are often 'incompatible', resulting in the loss of one of
them from the cell line. Therefore, plasmids can be assigned into
incompatibility groups, depending on their ability to coexist in a single cell.
These incompatibility groupings are due to the regulation of vital plasmid
functions.
An obvious way of classifying plasmids is by function. There are five
main classes:
 Fertility-(F)plasmids, which contain tra-genes. They are capable of
conjugation.
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 Resistance-(R)plasmids, which contain genes that can build a
resistance against antibiotics or poisons. Historically known as Rfactors, before the nature of plasmids was understood.
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 Col-plasmids, which contain genes that code for (determine the
production of) colicines, proteins that can kill other bacteria.
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 Degrative plasmids, which enable the digestion of unusual substances,
e.g., toluene or salicylic acid.
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 Virulence plasmids, which turn the bacterium into a pathogen.
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Plasmids can belong to more than one of these functional groups.
Plasmids that exist only as one or a few copies in each bacterium are,
upon cell division, in danger of being lost in one of the segregating bacteria.
Such single-copy plasmids have systems which attempt to actively distribute
a copy to both daughter cells.
Some plasmids include an addiction system. These plasmids produce both
a long-lived poison and a short-lived antidote. Daughter cells that retain a
copy of the plasmid survive, while a daughter cell that fails to inherit the
plasmid dies or suffers a reduced growth-rate because of the lingering poison
from the parent cell. This is an example of plasmids as selfish DNA.
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Applications of plasmids
Plasmids serve as important tools in genetics and biochemistry labs,
where they are commonly used to multiply (make many copies of) or
express particular genes. There are many plasmids that are commercially
available for such uses. Initially, the gene to be replicated is inserted in a
plasmid. These plasmids contain, in addition to the inserted gene, one or
more genes capable of providing antibiotic resistance to the bacterium that
harbors them. The plasmids are next inserted into bacteria by a process
called transformation, which are then grown on specific antibiotic(s).
Bacteria which took up one or more copies of the plasmid then express
(make protein from) the gene that confers antibiotic resistance. This is
typically a protein which can break down any antibiotics that would
otherwise kill the cell. As a result, only the bacteria with antibiotic resistance
can survive, the very same bacteria containing the genes to be replicated.
The antibiotic(s) will, however, kill those bacteria that did not receive a
plasmid, because they have no antibiotic resistance genes. In this way the
antibiotic(s) acts as a filter selecting out only the modified bacteria. Now
these bacteria can be grown in large amounts, harvested and lysed to isolate
the plasmid of interest.
Another major use of plasmids is to make large amounts of proteins. In
this case you grow the bacteria containing a plasmid harboring the gene of
interest. Just as the bacteria produces proteins to confer its antibiotic
resistance, it can also be induced to produce large amounts of proteins from
the inserted gene. This is a cheap and easy way of mass-producing a gene or
the protein it then codes for--for example, insulin or even antibiotics.
Plasmid Sex
.....In most bacteria there are several pieces of DNA. .. One is the somatic
genome - a huge circle of double-stranded DNA that actually measures
about 2 mm in length, and is all crammed into the little cell. ..This large
piece of DNA is what defines the type of bacterium it is. .. The cell cannot
live without this circle of DNA. In addition, there are various optional
smaller circles of DNA, which are usually called plasmids... To repeat:
..these are 'optional' and the cell can get along without them unless the genes
on those plasmids allow it to survive under unusual conditions such as when
a particular antibiotic is in the neighborhood, and the plasmid contains a
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gene that protects the cell from that antibiotic by any one of several different
mechanisms.
.....A cell can duplicate a plasmid and then send one copy over to another
cell via a thin tube called a pilus. ..Seemingly, each type of plasmid codes
for its own special type of pilus.
.....A widely used plasmid of E. coli is one called "F" (for fertility). .. Cells
that possess "F" are called male (F-donors or "F+"), these cells usually
possess two F-pili for the transport of the F into cells lacking it. .. Those
without F are called females (potential recipients, F-). .. In this exercise you
will mix a very few F+ with a much larger number of F-, and show that the Fcells are converted to F+ at a rather rapid pace. ..The rapidity is because
because it is a chain-reaction as the newly form F+ cells are added to the
pool of donors. ..(You can change this exercise into an experiment if you can
design an experiment from which you can determine the time it takes to
convert an F- into an F+.)
.....First, you must have a strain of donor that possesses a somatic genome
that is different from that of the recipient. ..That way you can "select
against" the original donor cells by using a mating medium in which they
cannot grow, but in which the recipients can. ..For this we will use donors
that are normal with respect to sensitivity to the antibiotic streptomycin,
while the recipients will be streptomycin resistant (str R). ..We will add
streptomycin to the medium, and none of the original donors will be able to
grow up to form colonies. ..(Always keep in the back of your mind a very
important word: "controls"!)
.....Second, you must have a way to tell whether or not the recipients have
acquired the F. ..We will do a little trick here, and use an F that has the
lactose operon inserted into it. ..This is called "F-lac." ..(Whenever other
genes are incorporated into F, the F is now called a type of F-prime (F').
..Thus F-lac is a type of F'. ..We will therefore start with F- cells that do not
have a functional lac-operon (lac-), and so when they acquire the F-lac, they
will be able to use lactose sugar to grow. .. (This is not a genetically
engineered product, but one that can be made naturally, and therefore doesn't
not need to be approved by an institutional recombinant DNA committee.
..Indeed, no part of this exercise is considered genetic engineering. ..It is all
genetic recombination using natural means. ..That is one of the important
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things about this exercise - this happens naturally around you all the time in
the genetically mobile and fluid microbial world.)
Figure 2: Transfer of Genetic Material through transformation,
translation, and conjugation
figure taken from Levy, 1998
see reference in text
Conjugation
Conjugation plays a large role in the spread of antibiotic resistance
through bacteria. This process involves direct cell-to-cell contact of two
bacterial cells, and the subsequent transfer of DNA. Conjugation can occur
between species that are unrelated; for this reason, a large gene pool is
available from which bacteria can exchange and acquire new genetic
material. (Guiney, 1984). Sex pili make contact between the donor and the
recipient cell. Once the two cell walls are in contact, this allows a mating
bridge to form. The plasmid DNA in the donor, possibly containing
antibiotic resistance genes, is nicked in one strand; this strand proceeds into
the recipient cell by undergoing rolling-circle replication (Hartl and Jones,
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1998, p316). Figure 3 demonstrates the process of rolling-circle replication.
Complementary copies of the DNA are produced in both the donor and the
recipient cells. Finally, the linear plasmid in the recipient becomes circular
and is ligated, and then both of the cells have a copy of the plasmid.
B. Conjugation - pilus transfers plasmid from one bacteria
to another
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Figure 3: Rolling Circle Replication
There are certain barriers to the process of conjugation, which often play a
role in the evolution of resistance. First, interactions at the cell surface are
involved. Contact must occur for mating to take place. Next, foreign DNA is
susceptible to host restriction modification systems which target and cleave
foreign DNA. Third, the new plasmid be unable to replicate in the new host.
An appropriate origin of replication in the new host may not be available,
thus preventing replication.
For example, transferred DNA is more likely to be stable in a new host if
it contains fewer restriction enzyme sites. This makes it less likely that the
DNA will be degraded by restriction enzymes which attack foreign DNA. A
selective advantage exists for plasmids with fewer restriction sites. Selective
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advantages which aid in stability can parallel the
beginnings of the evolution of strains that are resistant to
antibiotics. Antibiotic resistance will spread more readily
if the resistance genes can be transferred into non-resistant
cells. The ability to both invade many types of cells and to be maintained in
these cells are traits that are advantageous for survival and transfer of
plasmids.
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Transduction
Transduction occurs when a bacteriophage carries DNA from one species
to another. When a bacteriophage destroys its current host and invades a new
one, it may carry pieces of chromosomal DNA or plasmids from the
previous host. An occasional phage may carry some bacterial DNA.
Recombination can then occur between the phage (carrying bacterial DNA)
and the new host's bacterial DNA. Transfer of DNA thus occurs from one
bacterial cell to another, carried by a bacteriophage. ( Lacey, 1984 and Hartl
and Jones, 1998, p308). This provides another method for the spread of
resistance among bacteria.
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D. Transduction - a virus infects to bacteria carrying genetic
material form one bacteria to another
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What kind of genetic variation does this represent
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Transformation
Transformation is another method of acquiring resistance. During
transformation, bacterial cells take up DNA from the surrounding
environment. Certain requirements exist in order for transformation to take
place. First, exogenous DNA must be present in the immediate environment.
Bacteria must have mechanisms that allow the DNA to be taken up through
the bacterial cell walls. Also, the DNA must be incorporated into the
chromosome of the host, often by homologous recombination. During
homologous recombination, parts of the chromosome are replaced with
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related DNA (Maiden, 1998). Restriction modification systems play a role in
transformation as well as in conjugation. However, it is thought that since
these modification systems generate both DNA ends and smaller fragments,
restriction modification may actually increase the chance of recombination
with incorporated fragments. This could occur because recombination occurs
more frequently if the ends are homologous.
Transforming E. coli
Treatment of E. coli with the mixture of
religated molecules will produce some
colonies that are able to grow in the presence
of both ampicillin and kanamycin.
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A suspension of E. coli is treated with
the mixture of religated DNA
molecules.
 The suspension is spread on the
surface of agar containing both
ampicillin and kanamycin.
 The next day, a few cells — resistant
to both antibiotics — will have grown into visible colonies containing
billions of transformed cells.
Each colony represents a clone of transformed cells.
However, E. coli can be simultaneously transformed by more than one
plasmid, so we must demonstrate that the transformed cells have acquired
the recombinant plasmid.
Electrophoresis of the DNA from doubly-resistant colonies (clones) tells the
story.
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Plasmid DNA from cells that acquired their resistance from a
recombinant plasmid only show only the 3755-bp and 1875-bp
bands (Clone 1, lane 3).
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Clone 2 (Lane 4) was simultaneous transformed by religated pAMP
and pKAN. (We cannot tell if it took up the recombinant molecule as
well.)
Clone 3 (Lane 5) was transformed by the recombinant molecule as
well as by an intact pKAN.
C. Transformation - Pieces of DNA are absorbed into a competent cell
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What does this tell us about the virulence factor added
by the capsule?
Transformation with the Jellyfish Gene
Cloning other Genes
The recombinant vector described above could itself be a useful tool for
cloning other genes. Let us assume that within its kanamycin resistance
gene (kanr) there is a single occurrence of the sequence
5'GAATTC
3'
3' CTTAAG 5'
This is cut by the restriction enzyme EcoRI, producing sticky ends.
If we treat any other sample of DNA, e.g., from human cells, with EcoRI,
fragments with the same sticky ends will be formed. Mixed with EcoRItreated plasmid and DNA ligase, a small number of the human molecules
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will become incorporated into the plasmid which can then be used to
transform E. coli.
But how to detect those clones of E. coli that have been transformed by a
plasmid carrying a piece of human DNA?
The key is that the EcoRI site is within the kanr gene, so when a piece of
human DNA is inserted there, the gene's function is
destroyed.
All E. coli cells transformed by the vector, whether it
carries human DNA or not, can grow in the presence of
ampicillin. But E. coli cells transformed by a plasmid
carrying human DNA will be unable to grow in the
presence of kanamycin.
So,
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Spread a suspension of treated E. coli on agar
containing ampicillin only
 grow overnight
with a sterile toothpick transfer a small amount of each colony to an
identified spot on agar containing kanamycin
(do the same with another ampicillin plate)
Incubate overnight
All those clones that continue to grow on ampicillin but fail to grow on
kanamycin (here, clones 2, 5, and 8) have been transformed with a piece of
human DNA.
Some recombinant DNA products being used in human therapy
Using procedures like this, many human genes have been cloned in E. coli or
in yeast. This has made it possible — for the first time — to produce
unlimited amounts of human proteins in vitro. Cultured cells (E. coli, yeast,
mammalian cells) transformed with the human gene are being used to
manufacture:
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insulin for diabetics
factor VIII for males suffering from hemophilia A
factor IX for hemophilia B
human growth hormone (GH)
erythropoietin (EPO) for treating anemia
three types of interferons
several interleukins
granulocyte-macrophage colony-stimulating factor (GM-CSF) for
stimulating the bone marrow after a bone marrow transplant
granulocyte colony-stimulating factor (G-CSF) for stimulating
neutrophil production, e.g., after chemotherapy and for mobilizing
hematopoietic stem cells from the bone marrow into the blood.
tissue plasminogen activator (TPA) for dissolving blood clots
adenosine deaminase (ADA) for treating some forms of severe
combined immunodeficiency (SCID)
angiostatin and endostatin for trials as anti-cancer drugs
parathyroid hormone
leptin
hepatitis B surface antigen (HBsAg) to vaccinate against the
hepatitis B virus
Bacterial Transformation
This is a very basic technique that is used on a daily basis in a molecular
biological laboratory. The purpose of this technique is to introduce a foreign
plasmid into a bacteria and to use that bacteria to amplify the plasmid in
order to make large quantities of it. This is based on the natural function of a
plasmid: to transfer genetic information vital to the survival of the bacteria.
Bacterial Transformation and Transfection
Bacterial transformation is the process by which bacterial cells take up
naked DNA molecules. If the foreign DNA has an origin of replication
recognized by the host cell DNA polymerases, the bacteria will replicate the
foreign DNA along with their own DNA. When transformation is coupled
with antibiotic selection techniques, bacteria can be induced to uptake
certain DNA molecules, and those bacteria can be selected for that
incorporation. Bacteria which are able to uptake DNA are called
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"competent" and are made so by treatment with calcium chloride in the early
log phase of growth. The bacterial cell membrane is permeable to chloride
ions, but is non-permeable to calcium ions. As the chloride ions enter the
cell, water molecules accompany the charged particle. This influx of water
causes the cells to swell and is necessary for the uptake of DNA. The exact
mechanism of this uptake is unknown. It is known, however, that the
calcium chloride treatment be followed by heat. When E. coli are subjected
to 42degC heat, a set of genes are expressed which aid the bacteria in
surviving at such temperatures. This set of genes are called the heat shock
genes. The heat shock step is necessary for the uptake of DNA. At
temperatures above 42degC, the bacteria's ability to uptake DNA becomes
reduced, and at extreme temperatures the bacteria will die.
A Model of a Bacterial Plasmid
Legend:
Plasmids are similar to viruses, but lack a protein coat and cannot move
from cell to cell in the same fashion as a virus.
Plasmid vectors are small circular molecules of double stranded DNA
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derived from natural plasmids that occur in bacterial cells. A piece of DNA
can be inserted into a plasmid if both the circular plasmid and the source of
DNA have recognition sites for the same restriction endonuclease.
The plasmid and the foreign DNA are cut by this restriction endonuclease
(EcoRI in this example) producing intermediates with sticky and
complementary ends. Those two intermediates recombine by base-pairing
and are linked by the action of DNA ligase. A new plasmid containing the
foreign DNA as an insert is obtained. A few mismatches occur, producing an
undesirable recombinant.
The new plasmid can be introduced into bacterial cells that can produce
many copies of the inserted DNA . This technique is called DNA cloning.
The Recipe for Successful Interactions
First, it is necessary to construct the 'bait' and 'hunter' fusion proteins. The
'bait' fusion protein is the protein of interest (or 'bait') linked to the GAL4
binding domain, or GAL4 BD. This is done by inserting the segment of
DNA encoding the bait into a plasmid, which is a small circular molecule of
double-stranded DNA that occurs naturally in both bacteria and yeast. This
plasmid will also have inserted in it a segment of Gal4 BD DNA next to the
site of bait DNA insertion. Therefore, when the DNA from the plasmid is
transcribed and converted to protein, the bait will now have a binding
domain attached to its end (Figure 3). The same procedure is used to
construct the 'hunter' protein, where the potential binding partner is fused to
the GAL4 AD.
In addition to having the fusion proteins encoded for, these plasmids will
also contain selection genes, or genes encoding proteins that contribute to a
cell's survival in a particular environment. An example of a selection gene is
one encoding antibiotic resistance; when antibiotics are introduced, only
cells with the antibiotic resistance gene will survive. Yeast two-hybrid
assays typically use selection genes encoding proteins capable of
synthesizing amino acids such as histidine, leucine and tryptophan (Figure
4).
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Figure 4. Bait and Hunter Plasmids. The yeast two-hybrid
assay uses two plasmid constructs: the bait plasmid, which is
the protein of interest fused to a GAL4 binding domain, and
the hunter plasmid, which is the potential binding partner
fused to a GAL4 activation domain.
Once the plasmids have been constructed, they must next be introduced into
a host yeast cell by a process called "transfection". In this process, the outermembrane of a yeast cell is disturbed by a physical method, such as
sonification or chemical disruption. This disruption produces holes that are
large enough for the plasmid to enter, and in this way, the plasmids can cross
the membrane and enter the cell (Figure 5).
Once the cells have been transfected, it is necessary to isolate colonies that
have both 'bait' and 'hunter' plasmids. This is because not every cell will
have both plasmids cross their plasma membrane; some will have only one
plasmid, while others will have none. Isolation of transfected cells involves
identifying cells containing plasmids by virtue of their expressing the
selection genes mentioned previously. After the cells have been transfected
and allowed to recover for several days, they are then plated on minimal
media, or media that is lacking one essential nutrient, such as tryptophan.
The cells used for transfection are called auxotrophic mutants; these cells are
deficient in producing nutrients required for their growth. By supplying the
gene for the deficient nutrient in the 'bait' or 'hunter' plasmid, cells
containing the plasmid are able to survive on the minimal media, whereas
untransfected cells cannot (Figure 5). Selection in this way occurs in two
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rounds: first on one minimal media plate, to select for the 'bait' plasmid, and
then on another minimal media plate, to select for the 'hunter'4.
Once inside the cell, if binding occurs between the hunter and the bait,
transcriptional activity will be restored and will produce normal Gal4
activity. The reporter gene most commonly used in the Gal4 system is LacZ,
an E. coli gene whose transcription causes cells to turn blue4. In this yeast
system, the LacZ gene is inserted in the yeast DNA immediately after the
Gal4 promoter, so that if binding occurs, LacZ is produced. Therefore,
detecting interactions between bait and hunter simply requires identifying
blue versus non-blue.
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