Plasmids

Definition

Basically, plasmids are little, round particles of DNA that are fit for imitating autonomously. In
that capacity, they don't depend on chromosomal DNA of the life form for replication. Due to this
trademark, they are additionally alluded to as extra-chromosomal DNA. In spite of the fact that
the atom was first found in an individual from the Enterobacteriacae, thinks about have
demonstrated that plasmids are normally happening in numerous sorts of microorganisms around
the globe. In spite of the fact that there have been banters with respect to whether plasmids can be
viewed as microorganisms, in any event utilizing the proposed infection definition, it is important
that the expression "plasmid" is to a great extent used to allude to hereditary components that exist
outside the chromosome (in DNA of the living being) and are fit for imitating autonomously.

Plasmids are found in various organisms

 Bacteria
 Archaea
 Various eukaryotes (Yeast and plants)

Structure of plasmids

With respect to structure, plasmids are comprised of roundabout two fold chains of DNA. The
roundabout structure of plasmids is made conceivable by the two parts of the bargains strands
being joined by covalent bonds. The atoms are likewise little in size, particularly when contrasted
with the life forms' DNA, and measure between a couple kilobases and a few hundred kilobases.
Although a decent number of plasmids have a covalently shut round structure, a few plasmids have
a straight structure and don't frame a roundabout shape.

Components of Plasmids

Origin of replication

The origin of replication (ori) is a particular area in the strand at which replication starts. For
plasmids, this area is to a great extent made out of A-T base combines that are simpler to isolate
during replication.
Contrasted with the life forms' DNA that comprises of numerous sources of replication, plasmids
have one of a couple of roots of replication since they are littler in size. At the beginning of
replication, plasmids likewise contain various administrative components that add to the procedure
(for example Rep proteins)

Polylinker (multiple cloning sites)

In plasmid, the polylinker (MCS) is one of the most significant pieces of the particle. This is on
the grounds that it enables understudies to get familiar with cloning. Fundamentally, a polylinker
is a short grouping of DNA comprising of a couple of locales for cleavage by confinement
catalysts. All things considered, MCS takes into account simple inclusion of DNA through ligation
or confinement protein absorption. At the site of cleavage, distinctive polylinkers can cut the
strand. Accordingly, one of the limitation compounds can cut the plasmid at given purposes of the
sire to take into account DNA addition.

Antibiotic resistance gene

The anti-toxin opposition quality is one of the fundamental parts of plasmids. These qualities
assume a significant job in medication protection from (at least one anti-infection agents) therefore
making treatment of certain ailments all the more testing.

Plasmids are today known for their capacity to move starting with one types of microbes then onto
the next through a procedure known as conjugation (contact between cells that is trailed by
exchange of DNA content). All the while, they are equipped for presenting anti-microbial
opposition properties to different types of microscopic organisms.

While plasmid replication gives an additional favorable position to microbes (protection from
specific anti-microbials), it likewise influences cell division of microorganisms because of extra
replication trouble. Accordingly, microbes with plasmids will in general be out populated by those
without plasmids because of decreased cell division.
Types and functions of Plasmids

Resistance Plasmids

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:

a) Narrow-host-range group - Often replicated within a single species.

b) 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

In contrsat with different kinds of plasmids, degradative plasmids empower the host creature to
corrupt/separate xenobiotic mixes. Additionally, alluded to as headstrong substances, xenobiotic
mixes incorporate a scope of mixes discharged into the earth because of human activities and are
in this manner not normally happening or basic 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.
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:

i) Transposable element (IS2, 1S3, and Tn1000) ii) Replication sites (RepFIA, RepFIB,
and RepFIC) iii) 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. 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 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

Isolation of Plasmids

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

Applications of Plasmids

Humans have developed many uses for plasmids and have created software to record the DNA
sequences of plasmids for use in many different techniques. Plasmids are used in genetic
engineering to amplify, or produce many copies of, certain genes. In molecular cloning, a plasmid
is a type of vector. A vector is a DNA sequence that can transport foreign genetic material from
one cell to another cell, where the genes can be further expressed and replicated. Plasmids are
useful in cloning short segments of DNA. Also, plasmids can be used to replicate proteins, such
as the protein that codes for insulin, in large amounts. Additionally, plasmids are being investigated
as a way to transfer genes into human cells as part of gene therapy. Cells may lack a specific
protein if the patient has a hereditary disorder involving a gene mutation. Inserting a plasmid into
DNA would allow cells to express a protein that they are lacking.