2. JOSHUA LEDERBERG
WINS NOBEL PRIZE
The term plasmid was first introduced by
the American molecular biologist Joshua
Lederberg in 1952
Joshua Lederberg was an American
molecular biologist known for his work in
genetics , artificial intelligence , and space
exploration
He was just 33 years old when he won the
1958 Nobel Prize in Physiology
3. WHAT IS A PLASMID
* A plasmid is a small, circular, double-stranded
DNA * Plasmids naturally exist in
bacterial cells. * They also occur in
some eukaryotes.
Often, the genes carried in plasmids provide
bacteria with genetic advantages, such as antibiotic
resistance.
4.
5. IMPORTANCE OF PLASMID
Easy to work with - Plasmids are a convenient size (generally
1,000-20,000 basepairs) for physical isolation (purification) and
manipulation.
Self-replicatclonin - you can easily make an endless number
of copies of the plasmid using bacteria, which can uptake
plasmids and amplify them during cell division.
6. Stable
Plasmids are stable long-term either as purified DNA or within bacterial
cells that have been preserved as glycerol stocks.
7. PLASMID ELEMENT
1- origin of replication (ORI)
2-Antibiotic-resistance gene
3- At least one unique restriction enzyme recognition site.
4-Additionally, the restriction enzyme site(s)
9. Origin of Replication (ORI)
DNA sequence which directs initiation of plasmid replication
(by bacteria) by recruiting bacterial transcriptional machinery.
The ORI is critical for the ability of the plasmid to be copied
(amplified) by bacteria, which is an important characteristic of
why plasmids are convenient and easy to use.
10. 1- Allows for selection of plasmid-containing bacteria by
providing a survival advantage to the bacterial host.
2- Each bacterium can contain multiple copies of an individual
plasmid, and ideally would replicate these plasmids upon cell
division in addition to their own genomic DNA.
3- Bacteria without plasmids can replicate faster and out-
populate bacteria with plasmids, thus selecting against the
propagation of these plasmids through cell division
Antibiotic Resistance Gene
11. Multiple Cloning Site (MCS)
Short segment of DNA which contains several restriction enzyme
sites, enabling easy insertion of DNA by restriction enzyme
digestion and ligation.
In expression plasmids, the MCS is often located downstream
from a promoter, such that when a gene is inserted within the
MCS, its expression will be driven by the promoter.
As a general rule, the restriction sites in the MCS are unique and
not located elsewhere in the plasmid backbone.
12. Insert
The insert is the gene, promoter, or other DNA fragment
cloned into the MCS. The insert is typically the genetic
element one wishes to study using a particular plasmid.
13. Promoter Region
Drives transcription of the insert. The promoter is designed to
recruit transcriptional machinery from a particular organism or
group of organisms.
The promoter can also direct cell-specific expression.
14. Selectable Marker
The selectable marker is used to select for cells that have
successfully taken up the plasmid for the purpose of
expressing the insert.
This is different than selecting for bacterial cells that have
taken up the plasmid for the purpose of replication. The
selectable marker enables selection of a population of cells
that have taken up the plasmid and that can be used to study
the insert.
The selectable marker is typically in the form of another
antibiotic resistance gene (this time, under the control of a
non-bacterial promoter) or a fluorescent protein (that can be
used to select or sort the cells by visualization or FACS).
15. Primer Binding Site
A short single-stranded DNA sequence used as an initiation
point for PCR amplification or DNA sequencing of the
plasmid. Primers can be utilized to verify the sequence of the
insert or other regions of the plasmid
16. GENERAL TYPE OF PLASMID
Conjugative and Non-Conjugative
There are many ways to classify plasmids from general to
specific. 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.
17.
18. SPECIAL TYPES OF PLASMID
Fertility F-plasmids
Resistance plasmids
Virulence plasmids,
Degradative plasmids
Col plasmids
19. FERTILITY F-PLASMIDS
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.
20.
21. RESISTANCE PLASMIDS
Resistance 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. Recently, the type
bacterium that causes the sexually transmitted infection gonorrhea has
become so resistant to a class of antibiotics called quinolones that a new
class of antibiotics, called cephalosporins, has started to be recommended by
the World Health Organization instead. The bacteria may even become
resistant to these antibiotics within five years. According to NPR, overuse of
antibiotics to treat other infections, like urinary tract infections, may lead to the
proliferation of drug-resistant strains.
22.
23. VIRULENCE PLASMID
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.
24.
25. DEGRADITIVE PLASMID & COL
PLASMID
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.
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.
29. . The nucleoid is mostly composed of multiple compacted
copies of DNA in a continuous thread, with the addition of
some RNA and proteins. The DNA in prokaryotes is double-
stranded and generally takes a circular shape.
That DNA can sometimes also be found in other regions
outside the nucleoid.
Eukaryotes, such as plants and animals, have a nucleus that
houses their genetic material, with a surrounding double
membrane, or what we call the nuclear envelope. This
membrane separates the contents of the nucleus from the
cytoplasm. As in prokaryotes, the DNA of eukaryotes is also
double-stranded.
30. Bacterial chromosome
Chromosomes carry most prokaryotic genes. Genes essential for bacterial
growth are carried on chromosome. It is present in all bacteria. Only one
chromo-some is present in a bacterial nucleoid. Bacterial chromosome is a
single circle containing about 4000 kbp (kilobase pairs) of DNA. DNA is a
double stranded helical structure. A molecule of DNA consists of many
mononucleotides. A mononucleotide consists of a molecule of sugar, a
molecule of phosphate and a molecule of base. In each strand of DNA,
molecules of phosphate and sugar alternate, and one of the four bases
present in DNA is attached with the sugar. The four bases are adenine,
thymine, guanine and cytosine. A base of a strand joins with its
complementary base of the other strand. Adenine is always complementary to
thymine, and guanine to cytosine. During cell division, duplication of
chromosome occurs so that each daughter cell receives an identical set.
31. .
The bacterial chromosomal DNA is folded into a compact structure called
nucleoid. The shape and size of this ‘body’ is determined by a number of
factors. Major players are DNA supercoiling, macromolecular crowding and
architectural proteins, associated with the nucleoid
The early preparations of Varshavsky et al. revealed the presence of two
abundant proteins, H-NS (histone-like nucleoid structuring protein) and HU
(heat unstable protein)
32.
33.
34. REFERENCES
Hiraga, S. (1992). Chromosome and plasmid partition in Escherichia coli.
Annual Review of Biocheristry 61: 283–306.
Ippen-Ihier, K.A., Skurray, R.A. (1993). Genetic organization of transfer-
related determinants on the sex factor F and related plasmids, pp. 23–52.
In: Clewell, D.B. (ed.), Bacterial Conjugation. New York: Plenum Press.
