1. B.S.A. College
By- Arti Singh

2. Content
• Introduction
• What is Plasmid?
• Structure of Plasmid
• Plasmid Elements
• General properties of Plasmid
• Replication in Plasmid
• General types of Plasmid
• Specific types of Plasmid
• Other types of Plasmid
• Applications of Plasmid

3. Introduction
• The very existence of plasmids in bacterial
cytoplasm was revealed by Lederberg in 1952
while working on conjugation process in bacteria.
• Lederberg coined the term ‘Plasmid’ to refer to the
transmissible genetic elements that were
transferred from one bacterial cell to another and
determined the maleness in bacteria.
• Literally, thousands of plasmids are now known;
over 300 different naturally occurring plasmids
have been isolated from strains of Escherichia coli
alone.

4. What is Plasmid?
• A plasmid is a small, circular piece of DNA that is
different than the chromosomal DNA.
• They 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.
• Plasmids are mainly found in bacteria.
• Plasmids usually carry at least one gene and many of
the genes that plasmids carry are beneficial to their host
organisms.
• Although they have separate genes from their hosts,
they are not considered to be independent life.

7. General properties of Plasmid
• They are specific to one or a few particular bacteria.
• They replicate independently of the bacterial chromosome.
• They code for their own transfer.
• They act as episomes and reversibly integrate into bacterial
chromosome.
• They may pick-up and transfer certain genes of bacterial
chromosome.
• They may affect certain characteristics of the bacterial cell.
• Plasmids differ from viruses in following two ways-
They do not cause damage to cells and generally are
beneficial.
They do not have extracellular forms and exist inside cells
simply as free and typically circular DNA.

8. Replication in Plasmid DNA
There are three types of plasmid replication-
1. Rolling circle: Rolling circle replication mechanism is
specific to bacteriophage family m13 and the fertility F
factor which encodes for sex pili formation during
recombination by means of conjugation. Fragments
smaller than 10 kilo base usually replicate by this
replication mechanism as reported in some gram
positive bacteria. It allows the transfer of single stranded
replication product at a faster rate to the recipient cell
through pilus as in case of fertility factor or to the
membrane in case of phage.

10. 2. Col E1 type replication:
Col E1 replication is a negative
regulation mechanism which
enables the plasmid to control
its own copy numbers by
involving RNA type I, RNA type
II, Rom protein, and the
plasmid itself. Col E1
replication is initiated by means
of RNA-RNA interactions and
does not rely on replication
initiation protein encoded by
the plasmid to regulate its copy
number.

11. 3. Iteron-containing replicons:
This replicon consists of a gene that encodes Rep protein for
plasmid replication initiation, set of direct repeat sequences
called iteron, adjacent AT-rich region and Dna boxes which is a
protein required for bacterial chromosome replication initiation.
However length of adjacent AT-rich region and number of
iterons and DnaA boxes differs in a replicon.

12. General types of Plasmid
o Conjugative and Non-Conjugative
One way is by grouping them as either conjugative or non-conjugative.
Bacteria reproduce by sexual conjugation, which is the transfer of genetic
material from one bacterial cell to another, either through direct contact or
a bridge between the two cells. Some plasmids contain genes called
transfer genes that facilitate the beginning of conjugation. Non-conjugative
plasmids cannot start the conjugation process, and they can only be
transferred through sexual conjugation with the help of conjugative
plasmids.
o Incompatibility
Another plasmid classification is by incompatibility group. In a bacterium,
different plasmids can only co-occur if they are compatible with each
other. An incompatible plasmid will be expelled from the bacterial cell.
Plasmids are incompatible if they have the same reproduction strategy in
the cell; this allows the plasmids to inhabit a certain territory within it
without other plasmids interfering.

13. Specific types of Plasmid
There are five main types of plasmids:
1.Fertility Plasmids also known as F-plasmids, contain transfer genes
that allow genes to be transferred from one bacteria to another through
conjugation. These make up the broad category of conjugative
plasmids. F-plasmids are episomes, which are plasmids that can be
inserted into chromosomal DNA. Bacteria that have the F-plasmid are
known as F positive (F+), and bacteria without it are F negative (F–).
When an F+ bacterium conjugates with an F– bacterium, two
F+ bacterium result. There can only be one F-plasmid in each
bacterium
2.Resistance Plasmids or R plasmids contain genes that help a
bacterial cell defend against environmental factors such as poisons or
antibiotics. Some resistance plasmids can transfer themselves through
conjugation. When this happens, a strain of bacteria can become
resistant to antibiotics.

14. 3.Virulence Plasmids when a virulence plasmid is inside a bacterium, it
turns that bacterium into a pathogen, which is an agent of disease.
Bacteria that cause disease can be easily spread and replicated among
affected individuals. The bacterium Escherichia coli (E. coli) has several
virulence plasmids. E. coli is found naturally in the human gut and in other
animals, but certain strains of E. coli can cause severe diarrhea and
vomiting. Salmonella enterica is another bacterium that contains virulence
plasmids.
4.Degradative plasmids help the host bacterium to digest compounds that
are not commonly found in nature, such as camphor, xylene, toluene, and
salicylic acid. These plasmids contain genes for special enzymes that
break down specific compounds. Degradative plasmids are conjugative.
5.Col plasmids contain genes that make bacteriocins (also known as
colicins), which are proteins that kill other bacteria and thus defend the
host bacterium. Bacteriocins are found in many types of bacteria
including E. coli, which gets them from the plasmid ColE1.

15. Other types of Plasmid
Plasmids are versatile and can be used in many different ways by
scientists.
• Cloning Plasmids - Used to facilitate the cloning of DNA fragments.
Cloning vectors tend to be very simple, often containing only a bacterial
resistance gene, origin of replication, and MCS. They are small and
optimized to help in the initial cloning of a DNA fragment. Commonly used
cloning vectors include Gateway entry vectors and TOPO cloning vectors.
• Expression Plasmids - Used for gene expression (for the purposes
of gene study). Expression vectors must contain a promoter sequence, a
transcription terminator sequence, and the inserted gene. The promoter
region is required for the generation of RNA from the insert DNA via
transcription. The terminator sequence on the newly synthesized RNA
signals for the transcription process to stop. An expression vector can also
include an enhancer sequence which increases the amount of protein or
RNA produced. Expression vectors can drive expression in various cell
types (mammalian, yeast, bacterial, etc.), depending largely on which
promoter is used to initiate transcription.

16. • Gene Knock-down Plasmids - Used for reducing the
expression of an endogenous gene. This is frequently
accomplished through expression of an shRNA targeting the
mRNA of the gene of interest. These plasmids have promoters
that can drive expression of short RNAs.
• Genome Engineering Plasmids - Used to target and edit
genomes. Genome editing is most commonly accomplished
using CRISPR technology. CRISPR is composed of a DNA
endonuclease and guide RNAs that target specific locations in
the genome.
• Reporter Plasmids - Used for studying the function of genetic
elements. These plasmids contain a reporter gene (for example,
luciferase or GFP) that offers a read-out of the activity of the
genetic element. For instance, a promoter of interest could be
inserted upstream of the luciferase gene to determine the level of
transcription driven by that promoter.

17. • Viral Plasmids - These plasmids are modified viral
genomes that are used to efficiently deliver genetic material
into target cells. You can use these plasmids to create viral
particles, such as lentiviral, retroviral, AAV, or adenoviral
particles, that can infect your target cells at a high efficiency.

18. Applications of Plasmid
• 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.
• 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.
• In nature, plasmids often carry genes that benefit the survival of the
organism, such as by providing antibiotic resistance.

19. • Artificial plasmids are widely used as vectors in molecular cloning, serving
to drive the replication of recombinant DNA sequences within host
organisms.
• Plasmids are considered replicons, units of DNA capable of replicating
autonomously within a suitable host.
• This host-to-host transfer of genetic material is one mechanism
of horizontal gene transfer, and plasmids are considered part of
the mobilome.
• Unlike viruses, which encase their genetic material in a protective protein
coat called a capsid, plasmids are "naked" DNA and do not encode genes
necessary to encase the genetic material for transfer to a new host.
• However, some classes of plasmids encode the conjugative "sex"
pilus necessary for their own transfer.
• The size of the plasmid varies from 1 to over 200 kbp, and the number of
identical plasmids in a single cell can range anywhere from one to
thousands under some circumstances.
• The relationship between microbes and plasmid DNA is neither parasitic
nor mutualistic, because each impliesthe presence of an independent
species living in a detrimental or commensal state with the host organism.
• Plasmids can also provide bacteria with the ability to fix nitrogen.