Plasmid used as a vector

1. BY: PURVA R. DHAMANKAR
BSc. III MBB
SUBMITTEDTO: Dr.TUSHARWANKHEDE
Dr. DINESH KHEDKAR

2.  History
 Introduction
 Basic understanding of topic
 Process
 Applications
 Limitations
 Futuristic aspects
 Current status
 Conclusion
 References

3.  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 or Medicine for
discovering that bacteria can mate and
exchange genes. He shared the prize with
Edward L.Tatum and George Beadle who won
for their work with genetics.

4.  Recombinant DNA technology is a technique
used in genetic engineering that involves
identification, isolation and insertion of a gene
of interest in a vector such as plasmid to form a
recombinant molecule i.e. r-DNA and its
expression.
 The ultimate aim of these techniques is to
amplify the gene of interest and obtain its
product in large quantity
 The associated safety concerns make non-viral
delivery of therapeutic genes by using plasmid
DNA into cells is more attractive.

5.  A vector is a DNA molecule that has the ability
to replicate in an appropriate host cell and into
which the DNA fragment to be cloned is
integrated for cloning.
 Thus vector is an extra chromosomal DNA
autonomously replicating genetic element used
to carry a fragment of target DNA host cell for
the purpose of cloning and expression.
 It generally acts as a transporting vehicle for
foreign DNA from the test tube to host cell.

6. • A plasmid is a DNA molecule that is
separate from, and can replicate
independently of, the
chromosomal DNA.They are
double stranded and, in many
cases, circular. Plasmids usually
occur naturally in bacteria, but are
sometimes found in eukaryotic
organisms (e.g., the 2-micrometre-
ring in Saccharomyces cerevisiae).
 Plasmids are circular
deoxyribonucleic acid (DNA)
molecules that replicate
independently of the bacterial
chromosome.They are not
essential for the bacterium but
may confer a selective advantage

7.  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.
 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.

8. A schematic representation of
the pBR322 plasmid, one of the first
plasmids to be used widely as
a cloning vector. Shown on the
plasmid diagram are the genes
encoded
(amp and tet for ampicillin and tetrac
ycline resistance respectively), its
origin of replication (ori), and
various restriction sites (indicated in
blue).

9.  A plasmid is a vehicle that can carry
artificially inserted DNA. It will replicate in E.
coli, and with its own replication it will also
replicate the inserted DNA, independent of
it's origin. In a way one can see a plasmid as a
minute DNA factory.The main criteria for a
'good' plasmid is that it takes up the insert
you want to put in, and that it replicates in
sufficient amounts and that it does not
destroy your insert during the process.

10.  Replicate autonomously.
 Easy to replicate.
 Easily introduced in host cell.
 Easy to detect due to presence of
suitable marker gene.
 Able to integrate in host chromosome.
 Have small size less than 10kb.
 Growth is independent of the host’s cell
cycle amplification of gene product.

11.  ORI- the plasmid has its own replication and transcription mechanism for that it
has the DNA sequences that facilitate autonomous replication of plasmid DNA.
These sequences are called the origin of replication or ORI.
 Antibiotic resistance gene- the antibiotic resistance gene is one of the unique
properties of the plasmid.
 The antibiotic resistance gene present in plasmid helps the bacteria in survival
and protects them from the antibiotics.
 Interesting, the plasmid itself originates different antibiotic genes when it comes
in contact with different antibiotics.
 Restriction site- often known as multiple cloning sites, the plasmid also have
unique sequences that contain restriction digestion.The restriction site is used
for the insertion of a gene of interest during the experiment.
 For that, the plasmid is digested with the help of the unique restriction enzyme
having only a single digestion site on the plasmid.
 Promoter region- the promoter region facilitates transcription of target DNA.
 Selectable marker- the selectable marker allows selection of bacteria containing
the gene of interest.
 It is used in studying and manipulating the gene or the gene of interest.
 For performing any of the gene cloning experiments we must have to isolate
plasmid before constructing it.

23.  In DNA cloning, recombinant DNA molecules are formed in vitro by inserting
DNA fragments of interest into vector DNA molecules.The recombinant DNA
molecules are then introduced into host cells, where they replicate, producing
large numbers of recombinant DNA molecules that include the fragment of DNA
originally linked to the vector.
 The most commonly used cloning vectors are E. coli plasmids, small
circular DNA molecules that include three functional regions: (1) an origin of
replication, (2) a drug-resistance gene, and (3) a region where DNA can be
inserted without interfering with plasmid replication or expression of the drug-
resistance gene.
 Two enzymes are used to produce recombinant plasmids. Restriction enzymes
cut DNA at specific 4- to 8-bp sequences, often leaving self-
complementary single-stranded tails (sticky ends).These enzymes are used to
cut long DNA molecules into multiple restriction fragments and to cut
a plasmid vector at a single site. If a restriction fragment and cut plasmid vector
with complementary ends are mixed under the proper conditions, DNA ligase will
form phosphodiester bonds between the restriction fragment and vector DNA.

24.  When recombinant plasmids are incubated with E. coli cells first treated
with a high concentration of divalent cations , a very small fraction of the
cells take up a single recombinant plasmid.These transformed cells,
which carry the plasmid drug-resistance gene, can be selected by plating
on nutrient agar containing the antibiotic.All the cells in each colony that
grows on this medium contain identical plasmids descended from the
single plasmid that entered the founder cell of the colony. Isolated
colonies thus represent clones of the different restriction fragments
originally inserted into the plasmid vector.
 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.
 Single-stranded DNA containing up to 100 nucleotides of any desired
sequence can be chemically synthesized using automated instruments.
Synthetic dsDNAs are produced by synthesizing complementary ssDNAs
and then hybridizing them.

25.  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.
 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.Similarly, other biomolecules can also be produced.
 In addition, plasmids are used to administer gene therapy, which is a technique
used to correct defective genes responsible for disease development.They can
also be used to replicate proteins, such as the protein that codes for insulin, in
large amounts

27.  There are limitations to each of these steps:
large inserts require specialized plasmids
(cosmids orYACs for mega base sized- inserts),
the larger a plasmid + insert, the lower its
replication rate, but there are ways to improve
the yield, and certain plasmids result in frequent
deletions of (parts of) the insert, although this is
sometimes due to the host (the E. coli or another
host cell) or due to the nature of the insert as
well.

28.  We can assume that in future plasimds would
have wide applications in bioinformatics tools.
 Plasmid vectors can be used in production of
various drugs and medicines in order to treat
diseases.
 Plasmid vectors will have wide applications in
recombinant technology.
 Use of Plasmid vectors are likely to be involved
in the artificial evolution of recombinant
organisms.

29.  Prevention of disease:
 Using gene therapy techniques, single-gene disorders are now can
be prevented.
 Therapeutic drugs and proteins:
 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 transfer experiments:
 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.
 Besides this, the plasmid DNA is also used in gene mapping and
gene cloning as well.

30.  Plasmid DNA is one of the important element
in genetic research. Scientists are now trying
to use the viral vectors or other plasmid DNA-
mediated gene therapies for preventing the
disease like cystic fibrosis or Huntington’s
disease.
 However, the nonexpression behaviour of
plasmid and side effects of using viral vectors
are the two biggest challenges for scientists.

31.  Textbook of botany
 Biotechnology a problem approach 5th
addition.
 Wikipedia
 https://geneticeducation.co.in/plasmid-dna-
structure-function-isolation-and-
applications/
 Articles on internet.

32. Thank you!