PLASMID DNA PLASMUD VECTOR PLASMID PLASMID

Plasmid

1. •A multiple cloning site (MCS, or Polylinker region) is a DNA region
within a Plasmid that contains multiple unique Restriction enzyme
cut sites.
•Vector: an agent that can carry DNA into a cell or organism.
•cloning vector:
•An autonomously replicating genetic element used to carry a
cDNA or fragment of genomic DNA into a host cell for the purpose
of gene cloning. Commonly used vectors are bacterial plasmids
and modified bacteriophage genomes.
•DNA cloning:
•Recombinant DNA technique in which specific cDNAs or
fragments of genomic DNA are inserted into a cloning vector,
which then is incorporated into cultured host cells (e.g., E. coli
cells) and maintained during growth of the host cells; also called
gene cloning

2. •Plasmids Are Extrachromosomal Genetic
Elements
•Plasmids are extra pieces of genetic material
found in many cells that usually confer a specific
property to the cell.
•These properties include antibiotic resistance,
toxin production, and many other features.
•Plasmids are used in genetic engineering to
generate recombinant DNAs and as a mechanism
to transfer genes between organisms.

3. •Although plasmids are self-replicating
molecules (replicons) that reside within
host cells, they are not considered part
of the cell’s genome for two reasons.
•First, the same plasmid may exist in two
different species and be transferred
between these species.
•Second, some members of the same
species have plasmids, while others do
not.

4. •Plasmids are circular, double-stranded DNA
(dsDNA) molecules that are separate from a
cell’s chromosomal DNA.
•These extrachromosomal DNAs, which occur
naturally in bacteria, yeast, and some higher
eukaryotic cells, exist in a parasitic or
symbiotic relationship with their host cell.
•Plasmids range in size from a few thousand
base pairs to more than 100 kilobases (kb).

5. •some bacterial plasmids encode
enzymes that inactivate
antibiotics. Such drug-resistance
plasmids have become a major
problem in the treatment of a
number of common bacterial
pathogens

6. •Many of these plasmids also contain “transfer
genes” encoding proteins that can form a
macromolecular tube, or pilus, through which
a copy of the plasmid can be transferred to
other host cells of the same or related
bacterial species.
•Such transfer can result in the rapid spread of
drug-resistance plasmids, expanding the
number of antibiotic-resistant bacteria in an
environment such as a hospital

7. •The plasmids most commonly used in
recombinant DNA technology replicate in
E. coli.Generally, these plasmids have
been engineered to optimize their use as
vectors in DNA cloning.
• For instance, to simplify working with
plasmids, their length is reduced; many
plasmid vectors are only ≈3kb in length,
which is much shorter than in naturally
occurring E. coli plasmids.

9. •Most plasmid vectors contain little
more than the essential nucleotide
sequences required for their use
in DNA cloning:
•a replication origin, a
drug-resistance gene, and a
region in which exogenous DNA
fragments can be inserted

10. Here the selective gene is
amp
r
; it encodes the
enzyme β-lactamase,
which inactivates
ampicillin. Exogenous
DNA can be inserted into
the bracketed region
without disturbing the
ability of the plasmid to
replicate or express the
amp
r
gene.

11. Specific types of plasmids
•Plasmids can be divided into five main types:
•fertility F-plasmids,
•resistance plasmids,
•virulence plasmids,
• degradative plasmids, and finally
•Col plasmids

12. Fertility F-plasmids•Fertility plasmids, or
F-plasmids, are part
of a comprehensive
category of
conjugative plasmids
found in F+ or male
bacterial cells that
lead with frequent
transfer and rarely to
transfer of the
bacterial
chromosome

13. •F-plasmids can be inserted into
chromosomal DNA and are termed
episomes. Bacteria containing plasmid
are termed F positive (F+), and those
without plasmids are F negative (F–).
•Two F+ bacteria result upon conjugation
of an F+ bacterium with an F– bacterium.
Each bacterium is only able to contain
one F-plasmid

14. Resistance plasmids
•These plasmids contain antibiotics or poison
resistance genes and help bacterial production of
pili
•Resistance plasmids have the ability to use
conjugation to transfer themselves, thus providing
that bacterial strain resistance to antibiotics.
•The resistance to the antibiotics in bacteria may
even show within 5 years. That being said, NPR
stated that antibiotic overuse for treatment of
infections, such as UTIs, may result in the
appearance of drug-resistant strains.

15. Virulence plasmids
•A virulence plasmid containing bacterium will
render that bacterium a pathogen, or a
disease agent. Disease causing bacteria
easily spreads among individuals and causes
infection by replication in the new host.
•For example, there are several virulence
plasmids for the Escherichia coli bacterium.
•Salmonella enterica is additional example of a
virulence plasmid containing bacterium.

16. Degradative plasmids
•The main function of
degradative plasmids is to
aid the host bacterium in
the digestion of
compounds, which are not
commonly found in nature,
such as camphor, salicylic
acid, toluene, and xylene.
•Thus, these plasmids
encompass genes for
enzymes, which have a
main function to break
down specific compounds

Degradative plasmids can be described
as conjugative.

17. Col plasmids
•Col plasmids contain genes responsible for
production of bacteriocins also termed
colicins.
•These proteins can defend the host bacterium
through killing other bacteria.
•Numerous bacteria types produce
bacteriocins such as E. coli owing to the
presence of the ColE1 plasmid

18. Plasmid DNA Replication
•The replication origin (ORI) is a specific DNA sequence of
50 – 100 base pairs that must be present in a plasmid for
it to replicate. Host-cell enzymes bind to ORI, initiating
replication of the circular plasmid.
•Once DNA replication is initiated at ORI, it continues
around the circular plasmid regardless of its nucleotide
sequence
•Thus any DNA sequence inserted into such a plasmid is
replicated along with the rest of the plasmid DNA; this
property is the basis of molecular DNA cloning.

19. The parental strands are shown in
blue, and newly synthesized
daughter strands are shown in
red. The short segments represent
the A·T and G·C base pairs
connecting the complementary
strands.
Once DNA replication is initiated
at the origin (ORI), it continues in
both directions around the circular
molecule until the advancing
growing forks merge and two
daughter molecules are produced.

20. Application of plasmids
•Plasmids are used in genetic engineering to
amplify, or produce many copies of certain genes.
•They are used in different techniques and are
involved in research of genetic engineering and
gene therapy by gene transfer to bacterial cells or
to cells of superior organisms, whether other
plants, animals or other living organisms, to
improve their resistance to diseases, growth rates,
or any other required traits

21. •In molecular cloning, plasmids are types of
vectors that are useful in cloning short
segments of DNA. Scientists have developed
many uses for plasmids and have created
software to record the DNA sequences of
plasmids for the use in many different
techniques.
• For example, the artificial and cost-effective
bulk production of antibiotics can be
achieved by incorporating an expression
vector for that antibiotic in microbial cells.

22. Plasmids as genetic tools
In the molecular genetics, it is known as “vector” or
“construct” which helps in transferring the gene of
interest at the target location or we can say it is
used as a vehicle to transfer the gene of interest at
the specific location for artificial gene transfer or for
the development of the therapeutic proteins.

23. •The recombinant DNA method is also
used to transfer the gene for various
purposes such as for constructing GMO,
GMP and other resistance species of
plants.
•The plasmid DNA is also used in the gene
knockout study and construction of
knockout mice.
• the plasmid DNA is also used in gene
mapping and gene cloning as well

24. •One of the classic examples of the use of
plasmid or vector DNA in the
recombinant DNA technology is for the
production of insulin.
•Therapeutically important drugs and
proteins are synthesised artificially-
outside the cell using the plasmid DNA.
•Gene knockout method is used for constructing genetically
modified organism such as GM plants, GM bacteria and GM
animals. It is also used to study the effect and contribution of a
particular gene and its role in the development of a disease.

25. •Cloning plasmid:
•One of the simplest plasmid used in the
cloning experiment is cloning plasmid only
contains an antibiotic resistance gene, the
origin of replication and MCS.
•Viral plasmid:
•A modified viral genome is used as a viral
plasmid for delivering a gene of interest into
the host genome. The viral plasmid is
applicable in gene therapy experiments. AAV
and retrovirus are commonly used.

26. •Reporter plasmid:
•This type of plasmid is used to
study the function of a gene.
•Expression vector:
•This type of vector/ plasmid is
used to study the expression of a
gene of our interest.

27. Plasmid vectors containing a polylinker, or
multiple-cloning-site sequence, commonly are
used to produce recombinant plasmids carrying
exogenous DNA fragments
a. Sequence of a polylinker that
includes one copy of the
recognition site, indicated by
brackets, for each of the 10
restriction enzymes indicated.
Polylinkers are chemically
synthesized and then are inserted
into a plasmid vector.

28. •insertion of genomic restriction fragments into the pUC19
plasmid vector, which contains the polylinker
•One of the restriction enzymes whose recognition site is
in the polylinker is used to cut both the plasmid
molecules and genomic DNA, generating singly-cut
plasmids and restriction fragments with complementary
sticky ends (letters at ends of green fragments).
•By use of appropriate reaction conditions, insertion of a
single restriction fragment per plasmid can be
maximized. the restriction sites are reconstituted in the
recombinant plasmid.

29. •Polylinkers are synthetic oligonucleotides
composed of one copy of several different
restriction sites.
• Plasmid vectors that contain a polylinker will be
cut only once by multiple restriction enzymes,
each acting at its own site.
• Inclusion of a polylinker in a plasmid vector thus
permits cloning of restriction fragments
generated by cleavage of DNA with multiple
different restriction enzymes.