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1. Plasmids
Definition, Structure, Function, Vector and
Isolation
Definition: What are Plasmids?
Essentially, plasmids are small, circular molecules of DNA that are capable of replicating
independently. As such, they do not rely on chromosomal DNA of the organism for
replication. Because of this characteristic, they are also referred to as extra-
chromosomal DNA.
Although the molecule was first discovered in a member of the Enterobacteriacae,
studies have shown that plasmids are naturally occurring in many types
of microorganisms around the world.
* Although there have been debates as to whether plasmids can be regarded as
microorganisms, at least using the proposed virus definition, it is worth noting that the
term "plasmid" is largely used to refer to genetic elements that exist outside the
chromosome (in DNA of the organism) and are capable of replicating independently.
Plasmids can be found in:
 Bacteria
 Archaea
 Various eukaryotes (yeast and plants)
Bacterium with its chromosomal DNA and several
plasmids within it. User:Spaully on English wikipedia [CC BY-SA 2.5
(https://creativecommons.org/licenses/by-sa/2.5)]
Structure

2. With regards to structure, plasmids are made up of circular double chains of DNA. The
circular structure of plasmids is made possible by the two ends of the double strands
being joined by covalent bonds. The molecules are also small in size, especially when
compared to the organisms' DNA, and measure between a few kilobases and several
hundred kilobases.
Although a good number of plasmids have a covalently closed circular structure, some
plasmids have a linear structure and do not form a circular shape.
Generally, plasmids are composed of three major components that include:
Origin of replication (replicon) - The origin of replication (ori) refers to a specific
location in the strand at which replication begins. For plasmids, this location is largely
composed of A-T base pairs that are easier to separate during replication.
Compared to the organisms' DNA that consists of many origins of replication, plasmids
have one of a few origins of replication because they are smaller in size. At the origin of
replication, plasmids also contain a number of regulatory elements that contribute to
the process (e.g. Rep proteins)
Polylinker (multiple cloning sites) - In plasmid, the polylinker (MCS) is
one of the most important parts of the molecule. This is because it allows
students to learn more about cloning. Basically, a polylinker is a short
sequence of DNA consisting of a few sites for cleavage by restriction
enzymes.
As such, MCS allows for easy insertion of DNA through ligation or restriction
enzyme digestion. At the site of cleavage, different polylinkers can cut the
strand. Therefore, one of the restriction enzymes can cut the plasmid at
given points of the sire to allow for DNA insertion.
Antibiotic resistance gene - The antibiotic resistance gene is one of the
main components of plasmids. These genes play an important role in drug
resistance (to one or more antibiotics) thus making treatment of some
diseases more challenging.
Plasmids are today known for their ability to transfer from one species of
bacteria to another through a process known as conjugation (contact
between cells that is followed by transfer of DNA content). In the process,
they are capable of conferring antibiotic resistance properties to other
species of bacteria.
* While plasmid replication provides an added advantage to bacteria
(resistance to certain antibiotics), it also affects cell division of bacteria due
to additional replication burden. As a result, bacteria with plasmids tend to
be out populated by those without plasmids due to reduced cell division.
Some of the other components of plasmids include:

3. A promoter region - This is the component of plasmids that is involved in recruiting
transcriptional machinery
Primer binding site - This is a short sequence of DNA on a single strand that is
typically used for the purposes of PCR amplification or DNA sequencing
Although plasmids share various general characteristics, there are different types in
existence.
Comparing the
activity of non-integrating plasmids, on the top, with episomes, on the bottom, during cell
division by Spaully [CC BY-SA 2.5 (https://creativecommons.org/licenses/by-sa/2.5)]
Types and Functions of Plasmids
Resistance Plasmids

4. Also referred to as antimicrobial resistance plasmids, resistance plasmids are a type of
plasmids that carry genes that play an important role in antibiotic resistance. They are
also highly involved in bacterial conjugation by producing conjugation pili which transfer
the R plasmid from one bacterium to another.
Resistance plasmids are divided into two main groups that include:
Narrow-host-range group - Often replicated within a single species.
Broad-host-range group - Easily transferred between bacteria species. This group of
resistance plasmids has been shown to carry a range of antibiotic resistance genes.
Following the transfer of antibiotic resistance genes to drug-sensitive bacteria, this can
cause the bacteria to develop resistance towards a variety of drugs.
Degradative Plasmids
Compared to other types of plasmids, degradative plasmids enable the host
organism to degrade/break down xenobiotic compounds. Also referred to as
recalcitrant substances, xenobiotic compounds include a range of compounds
released into the environment as a result of human actions and are therefore
not naturally occurring or common in nature.
Hosts of degradative plasmids are found in groups IncP-1, IncP-7, and IncP-
9 and include such species as
Ochrobactrum anthropi, Rhizobium sp, Burkholderia hospita, Escherichia coli,
and Pseudomonas fluorescens among many others.
Because of the ability of the host to degrade xenobiotic compounds,
researchers have attempted to use the plasmids to degrade various
contaminating substances in the environment. However, given that this has
not proved effective, research studies continue to be conducted to determine
how to use various indigenous bacteria (as hosts of degradative plasmids)
for degradation of such compounds.
While degradative plasmids contribute to the degradation of xenobiotic
compounds, their behavior varies depending on a number of factors such as
the capacity for replication and stability. For instance, plasmids found
in IncP-1 group have not only been shown to have a broad host range, but
also high transfer frequency.

5. The differences in the behavior of different degradative plasmids have
therefore been shown to result in different behaviors between them and
their respective hosts.
* The use of biodegradative microorganisms for the purposes of removing
xenobiotic compounds from contaminated environments is known as
Bioaugmentation.
* Whereas IncP-1 plasmids have a broad range of hosts, IncP-7 has been
shown to have a narrow host range. On the other hand, IncP-9 has an
intermediate host range.
Fertility Plasmids
Like many other plasmids, fertility plasmids (F plasmid) have a circular structure and
measures about 100 kb.
Some of the main parts of the F plasmid include:
 Transposable element (IS2, 1S3, and Tn1000)
 Replication sites (RepFIA, RepFIB, and RepFIC)
 Origin of conjugative transfer (oirT)
 Replication origin regions
F plasmid plays an important role in reproduction given that they contain genes that
code for the production of sex pilus as well as enzymes required for conjugation. F
plasmid also contains genes that are involved in their own transfer. Therefore, during
conjugation, they enhance their own transfer from one cell to another.
Whereas the cells that process the F plasmids are referred to as donors, those that lack
this factor are the recipients. On the other hand, the plasmids that enhance the ability
of the host cell to behave like a donor are known as the transfer factor.
During conjugation, the donor cell (bacteria) with sex pili (1-3 sex pili) binds to a
specific protein on the outer membrane of the recipient thus initiating the mating
process.
Following the initial binding, the pili retract thus allowing the two cells to bind together.
This is then followed by the transfer of DNA from the donor to the recipient and
consequently the transfer of the F plasmid. As a result, the recipient acquires the F
factor and gains the ability to produce sex pilus involved in conjugation.
* During conjugation, only DNA is passed from the donor to the recipient. Therefore,
cytoplasm and other cell material are not transferred.

6. * Sexual pili (sex pili) are tiny rod-like structures that allow the F-positive (cells that
have the F factor) bacterial cells to attach to the F-negative (cells lacking the pili)
female to promote conjugative transfer.
Col Plasmids
Col plasmids confer to bacteria the ability to produce toxic proteins known as colicines.
Such bacteria as E. coli, Shigella and Salmonella use these toxins to kill other bacteria
and thus thrive in their respective environments.
There are different types of Col plasmids in existence that produce different types of
colicines/ colicins. A few examples of Col plasmids include Col B, Col E2 and E3. Their
differences are also characterized by differences in their mode of action.
For instance, whereas Col B causes damage to cell membrane of other bacteria (lacking
the plasmid) Col E3 has been shown to induce degradation of the nucleic acids of the
target cells.
Like fertility plasmids, some of the Col plasmids have been shown to carry elements
that enhance their transmission from one cell to another. Therefore, through
conjugation or the mating process, particularly for cells with the F factor (fertility
plasmids) the Col plasmids can be transferred from one cell (donor) to another
(recipient). As a result, the recipient acquires the ability to produce toxins that kill or
inhibit the growth of the target bacteria lacking the plasmid.
* Colicins/colicines belong to a group of toxins known as bacteriocins.
* These toxins affect the target bacteria by affecting such processes as replication of
DNA, translation and energy metabolism among others.
Virulence Plasmids
Compared to other harmless bacteria, bacteria that tend to be pathogenic in nature
carry genes for virulence factors that allow them to invade and infect their respective
hosts.
For some of these bacteria, the virulence factors are the result of the organisms' own
genetic material. However, for others, this is as a result of genetic elements from extra-
chromosomal DNA. Although there are other sources of such elements, e.g.
transposons, plasmids are some of the most common mobile genetic elements.
With regards to pathogenicity, virulence plasmids play an important role given that they
can help bacteria effectively adapt to their respective environments. This is because the
virulence plasmid can enable the organism to express an array of virulence-associated

7. functions thus providing the organism with more advantageous characteristics to thrive
in their environment.
Like other types of plasmids, virulence plasmids can also be transmitted from one
bacterium to another. Apart from the virulence gene, plasmids have also been shown to
carry other important elements that enhance transmission and maintenance.
For this reason, they are larger in size but low in numbers. This ensures that they do
not cause additional burden to the organism during cell division.
Typically, cell division and cell maintenance require the use of energy. By having low
numbers of virulence plasmids, the cells are spared significant metabolic burden that
would be required for maintenance and genome duplication of numerous plasmids.
Some of the other types of plasmids include:
 Recombinant plasmids - Plasmids that have been altered in the laboratory and
introduced into the bacteria for the purposes of studies
 Crptic plasmids - No known functions
 Metabolic plasmids - Enhance metabolism of the host
 Conjugative plasmids - Promote self-transfer
 Suicide plasmids - Fail to replicate when transferred from one cell to another
Plasmid Vector
A vector refers to any piece of molecule that contains genetic material that can be
replicated and expressed when transferred into another cell. Based on this definition, it
is possible to see why the words "vector" and "plasmids" are sometimes interchanged.
However, this is not to say that all plasmids are vectors.
One of the primary characteristics of plasmid vectors is that they are small in size.
Apart from their size, they are characterized by an origin of replication, a selective
marker as well as multiple cloning sites.
The ideal plasmid vectors have high copy numbers inside the cell. As such, it ensures
high numbers of the target gene for cloning purposes. This also ensures that the gene
of interest is increased during genomic division. In addition, the plasmid can have a
marker gene as the visual marker to help determine whether cloning was successful.
Because of their multiple cloning sites, plasmids have been shown to be some of the
best vectors for cloning. Because of this characteristic, it is possible for restriction
enzymes to cleave various regions of the plasmid for cloning.
Over the years, using these vectors has allowed for recombinant DNA to be introduced
into host cells for study purposes. For instance, through this type of cloning, it has

8. become possible for researchers to sequence the genome of a range of species, study
the expression of genes and even observe various cellular mechanisms.
* While smaller plasmids are capable of carrying long DNA segments, reduced size can
also help remove non-essential genes that are not required for cloning.
Plasmid Isolation
In order to obtain purified plasmid DNA for such procedures as cloning, PCR and
transfection, plasmid isolation has to be performed. The process involves using a
number of techniques to obtain the plasmid DNA from host cells in order to use it in
molecular biology.
Plasmid isolation involves the following steps:
Cell growth (growth of bacterial cells) - This involves growing the bacteria that
contain plasmid in a specific shaken culture. Here, given antibiotics may be used to
prevent the growth of other undesired bacteria.
Centrifugation - Bacterial growth is followed by centrifugation in order to pellet the
cells. Once the supernatant has been removed, then isolation of plasmids can begin.
One of the most common techniques for isolation is the classical method that is
sometimes referred to as alkaline lysis.
This involves the following steps:
 Suspension of the pellet in an isotonic solution - The bacterial pellet obtained
from centrifugation is re-suspended in an isotonic solution (ethylene
diamine tetraacetate) which prevents nuclease activity
 Alkaline lysis of the cells - This involves cell lysis by using sodium dodecyl sulfate
to disintegrate the lipid structure on the cell membrane
 Precipitation of dissolved proteins using a solution of acidic potassium acetate
 Sedimentation - centrifugation is used for sedimentation
 Purification - A mixture of phenol and chloroform is used for purification of the
plasmid DNA. This step removes protein content
 Add ethanol for precipitation (to be sedimented through centrifugation)
 Wash the solution with 70 percent ethanol (to remove salt content)
 Centrifuge to sediment plasmid DNA
 Dissolve in TE solution and store