medical education

1. PLASMID AND ITS IMPORTANCE

2. Introduction
 Plasmids are extrachromosomal , autonomously replicating
genetic elements
 Covalently closed, circular dsDNA, ranging from size < 10 to
> 400 kb pairs
 Naturally occurs in bacteria, may also be found in other
eukaryotes

3.  Stably inherited and exchanged between a broad spectrum of
bacteria
 Plasmids allow bacterial populations to ‘sample’ the horizontal
gene pool for adaptive traits that might be advantageous for
survival under local selective pressure
 Can serve as a vehicle that can carry artificially inserted DNA,
self replicate and also replicate the inserted DNA
 Provide genetic variation, act as sources of recombination and
can allow faster gene fixation leading to greater likelihood that
the ‘new’ trait will persist

4. History
 1903- Walter S. Sutton and Theodor Boveri proposed hereditary
units on chromosomes.
 1910-1950- Extensive research was done based on the above theory.
Numerous studies on E.coli mating and relationship with
chromosomes.
 1952- Joshua Lederberg suggested the name “Plasmid”
 1958 – Lederberg won the Nobel Prize in Physiology and Medicine
 1962- Circular DNA was found
 Modern Research- Used in DNA replication(cloning),also used to
genetically alter organisms with recombinant DNA

5. Structure of a plasmid Plasmids have 3 key parts-
 The origin of replication is
used to indicate where DNA
replication is to begin.
 The selectable marker gene is
used to distinguish cells
containing the plasmid from
cells that don’t contain it.
 The cloning site is a site in the
plasmid where the DNA is
inserted.

6.  Some plasmids encode a protein involved in the initiation of
replication, termed Rep protein and other genes involved in
replication control.
 Restriction Enzyme sites – in the non-essential regions of the plasmid
enable cutting and pasting of DNA into a plasmid
 tra encoded proteins necessary for replication
 The LacZ gene produces Beta-galactosidase. Also acts as a selectable
marker used for detection of cells containing plasmids. (Beta-
galactosidase action results in a blue by-product when X-gal is
present in the growth medium).

7. •Recombinant
plasmid containing
colonies-blue
•Non recombinant
plasmid containing
colonies -white

8. Classification of plasmids
I. Based on ability to undergo conjugation
II. Based on function
III. Based on shape
IV. Based on replication control
V. Based on Replicating Mechanisms

9. I. BASED ON ABILITY TO UNDERGO CONJUGATION:
1. Conjugative plasmids: Able to perform conjugation, which
is the process of transferring plasmids to another environment
2. Non-conjugative plasmids: Unable to perform conjugation
3. Intermediate plasmids (Mobilizable): Able to be transferred
by conjugative plasmids. They contain a subset of genes

10. Conjugation - by Lederberg and Tatum (1946) – E.coli (K12)
 Process by which ‘male’ or ‘donor’ bacterium mates or makes
physical contact with a ‘female’ or ‘recipient’ bacterium and
transfers genetic elements in to it.
 Donor cell – Plasmid encodes sex pilus (Projects from cell
surface)
 Pilus comes in contact with recipient cell
 Plasmid DNA replicates and passes copy of it from donor to
recipient along sex pilus – called Conjugation tube
 Recipient now capable of further conjugation

12. II. BASED ON THEIR FUNCTION:
1. Fertility plasmids (F plasmid): can perform conjugation
2. Resistance plasmids (R plasmid): contain genes that encode
resistance against antibiotics or poisons
3. Colcinogenic plasmids (Col plasmid): contain genes that code for
proteins that can kill bacteria (bacteriocins)
4. Degradative plasmids: enable digestion of unusual substances
(toluene and salicylic acid )
5. Virulence plasmids (Ti plasmid): turn the host of the plasmid into
a pathogen by causing production of opines,toxins etc.
6. Cryptic plasmid : serves no function

13. F Plasmid :
 F factor - Transfer factor containing
genetic information for synthesis of
sex pilus
 F+ cells mate with F- cells and render
them F+

14. R Plasmid :
 Resistance transfer Factor (RTF):
Responsible for spread of multi drug resistance
 Resistance determinant (r) :
Present for each of the several drugs, resistance to 8 or more
drugs transferred simultaneously

15. Col plasmid :
 Colicin produced by Coliform bacteria – determined by Colcinogenic
plasmid containing the Col factor
 Colicins are protein produced by some strains of Escherichia coli and
Shigella
 They exert cytotoxic effect by depolarisation of the cytoplasmic
membrane, have DNase activity, RNase activity, or inhibit murein synthesis
 They have a bactericidal effect on the same species or closely related
bacteria, but have no effect on the strains which produce them
 Each Col plasmid confers immunity to the particular type of colicin which
it encodes

16.  Colicin typing is used
- to show the epidemiological relation in E. coli and Shigella
infections.
-as a basis for differentiating and identifying
epidemiologically related strains in outbreaks

17. III. BASED ON SHAPE:
1. Nicked Open-Circular: DNA has one strand cut
2. Relaxed Circular: DNA is fully intact
3. Linear: DNA has free ends (Borrelia,Mycobacteria)
4. Supercoiled: DNA is fully intact but has a twist in it, making
it more compact
5. Supercoiled Denatured: Slightly less compact than
supercoiled

18. IV. BASED ON REPLICATION CONTROLS:
1. Incompatible plasmids: Two plasmids are incompatible if
either is less stable in the presence of the other than it was by
itself, but they have the same replication control genes
2. Compatible plasmids: Plasmids are compatible if they can
coexist and replicate within the same bacterial cell, however
have different replication controls

19. V. BASED ON REPLICATING MECHANISMS:
1. Rolling circle replicating plasmids
2. Theta replicating plasmids
3. Strand displacement replicating plasmids

20. 1. Rolling circle replicating plasmids

21. 2.Theta replicating plasmids
•Widespread among plasmids
from gram negative bacteria,
also seen in some gram
positive plasmids.
•Electron Microscopy shows
that replicating intermediates
appear as bubbles (early
stages) that, when they
increase in size, result in
theta-shaped molecules.

22. 3.Strand displacement replicating plasmids
•Replication occurs from two
symmetrical and adjacent single-
stranded origins positioned one on
each DNA strand.
•Synthesis of each one of the
strands occurs continuously and
results in the displacement of the
complementary strand.
•Replication of this displaced
strand again occurs

23. Role & uses of
Plasmids

24.  Mediators of antibiotic resistance, heavy metal resistance, virulence
 Also have environmental adaptability and persistence, metabolic
functions
 Cause biodegradation of toxic substances- toluene, hydrocarbons,
herbicides and pesticides
 Plasmid profiling has a role in disease outbreak analysis
 Act as cloning vectors
 Plasmid Isolation techniques provide ready to use DNA for genetic
engineering
 Plasmid Vaccines

25. Role in virulence

26. Role in metabolic activities
Pseudomonas species Degradation of camphor, toluene, octane, salicylic acid
Bacillus
stearothermophilus
Degradation of Amylase
Alcaligenes eutrophus Utilization of H2 as oxidizable energy source
Escherichia coli Sucrose uptake and metabolism, citrate uptake
Klebsiella species Nitrogen fixation
Streptococcus (group N) Lactose utilization, galactose phosphotransferase system, citrate
metabolism
Rhodospirillum rubrum Synthesis of photosynthetic pigment
Flavobacterium species Nylon degradation

27. Role in drug resistance
GENE RESISTANCE
CONFERRED TO
ENCODED PROTEIN MODE OF ACTION
Cm Chloramphenicol Acetyl transferase Acetylates the drug
Sm Streptomycin -Adenylate transferase
-Phosphotransferase
-Adenylates
-Phosphorylates
Sp Spectinomycin Adenylate transferase Adenylates
Tc Tetracycline Tet proteins Excludes drug from
cell

28. GENE RESISTANCE
CONFERRED
TO
ENCODED PROTEIN MODE OF
ACTION
dfrA Trimethoprim dihydrofolate
reductase (DHFR)
altered DHFR
enzymes
qnr Quinolone DNA gyrase Alteration of
enzymes
Ap Ampicillin ß lactamase Hydrolyses
mel Erythromycin permease Hydrolyses lactone
ring
van A Vancomycin d-alanine-d-alanine Alters the cell wall

29. Plasmid Profiling
 Plasmid profiling or plasmid fingerprinting is a technique of isolation of
plasmids present in a bacterial cell
 Done by simple cell lysis followed by agarose gel electrophoresis
 Plasmid counts and sizes are the basis for strain identification
 Used in analysis of outbreaks of Nosocomial and community acquired
infections, trace inter- and intra-species spread of antibiotic resistance
 Most strains are typeable and have good ease of interpretation
 Reproducibility of this method is difficult as the plasmid exists in different
forms – supercoiled, nicked, linear – They exhibit different profiles
 Clinical isolates lacking plasmids are untypable

31. Plasmids In Genetic
Engineering

33. Plasmid Isolation Techniques
 For plasmid isolation, bacterial cultures are grown to late
logarithmic/early stationary phase
 Plasmids usually occur in the covalently closed circular (supercoiled)
configuration within host cells
 Gentle cell lysis eliminates intracellular macromolecules, and
plasmid DNA is enriched and purified.

34.  Materials required : Antibiotic (to maintain the plasmid. Only
bacteria which take up copies of the plasmid survive the
antibiotic, since the plasmid makes them resistant), Tris+
EDTA buffer (Tris – alkaline buffer for DNA; EDTA –
chelator)
 For long-term storage, plasmid DNA should be frozen in
aliquots of storage TE buffer
 Repeated thawing and freezing of DNA should be avoided.

35. Various Methods of Isolation
1. Rapid boiling method for small plasmids in E. coli (Centrifuged
culture is mixed with Sucrose + Tris EDTAagarose gel
electrophoresis)
2. Hot alkaline method for all plasmid sizes and bacteria
(Centrifuged Culture mixed with Tris EDTA Hot samples of
Phenol/Chloroform(1:1) is addedAGE)
3. Lysozyme method for various Gram-negative bacteria
4. Lysis of cells from single colonies on agarose gel
5. Plasmid isolation from Gram-positive bacteria, especially
lactobacilli, with mutanolysin or lysozyme
6. Lysis of Gram-positive bacteria with lysostaphin

36.  The amplified DNA will subsequently be cloned in a plasmid
vector using a cloning technique
 Ultra pure, ready to use pDNA
 The isolated pDNA is ready to use in downstream
applications.

37. Applications of Plasmids in Genetic Engineering
 Plasmids are extremely valuable tools in the fields of molecular
biology and genetics, specifically in the area of genetic engineering
 Plasmids are simple to construct and easily propagated in large
quantities
 Possess an excellent safety profile, with virtually no risk of
oncogenesis (as genomic integration is very inefficient) and
relatively little immunogenicity
 Plasmids have a very large DNA packaging capacity and can
accommodate large segments of genomic DNA

38.  They are easy to handle, remaining stable at room temperature for
long periods of time (an important consideration for clinical use)
 The main limitation with plasmids is poor gene transfer efficiency
 Given the potential benefits, plasmid-mediated gene therapy
represents an attractive option in many respects
 They play an important role in:
 Gene cloning
 Recombinant Protein production (eg. human insulin, growth
hormone) and Gene Therapy

39. Plasmids in Gene Cloning
 Used as a cloning vector to carry a gene not found in the
bacterial host chromosome
 In DNA cloning, rDNA molecules are formed in vitro by
inserting DNA fragments of interest into vector DNA
molecules. The rDNA molecules are then introduced into host
cells, where they replicate, producing large numbers of rDNA
molecules that include the fragment of DNA originally linked
to the vector.
 The most commonly used cloning vectors are E. coli plasmids
 Small synthetic DNA can also be incorporated into the plasmid
vector

41. Applications of Gene Cloning
 Production of recombinant protein (GH, Insulin, other growth
factors, interleukins/cytokines)
 Recombinant vaccines
 Identification of genes responsible for human diseases
(mapping the breast cancer gene BRCA1)
 Gene therapy (Somatic-therapeutic genes are tranferred into
somatic cells of patient and Germline therapy-germ cells are
modified by introducing functional genes into their genome)

45. Plasmid (DNA) vaccines
 Since its early applications in the 1950's, DNA-based immunization
using recombinant DNA technology has become a novel approach to
vaccine development
 Direct injection of naked plasmid DNA induces strong immune
responses to the antigen encoded by the gene vaccine
 Once the plasmid DNA construct is injected host cells take up the
foreign DNA, expressing the gene  produce the corresponding
protein inside the cell.
 Induces both MHC class I and class II restricted cellular and humoral
immune responses

46.  Once constructed, the vaccine
plasmid is transformed into
bacteria where bacterial
growth produces multiple
plasmid copies  plasmid
DNA is then purified from the
bacteria (separate the circular
plasmid from the much larger
bacterial DNA and other
bacterial impurities)
 This purified DNA acts as the
vaccine

48. Routes of administration of DNA vaccines:
 Subcutaneous or intradermal injection
 Topical: transfects Langerhans cells via a dermal patch that consists
of a nanoparticle that carries antigen-encoding plasmid DNA
 Painting DNA: consists of stripping a few layers of the skin in order
to acheive a more successful transfection.
 Assisted by a vesicular system: involves the application of a vaccine
to intact skin facilitated by a carrier (liposomes, niosomes,
transfersomes)

49.  Other routes- Aerosol instillation of naked DNA
on nasal and lung mucosa
- topical administration of pDNA to the eye and
vaginal mucosa.
-biodegradable microspheres attenuated Shigella
or Listeria vectors for oral administration to the intestinal
mucosa, and recombinant adenovirus vectors

50. Gene gun delivery :
•Plasmid DNA (pDNA) that has
been adsorbed onto gold
or tungsten microparticles is
bombarded into the target cells
using compressed helium as an
accelerant
•Langerhans cells and
keratinocytes become directly
transfected by the bombardment
of gold particles coated with DNA

51. Advantages of DNA vaccines:
 Induces the expression of antigens that resemble native viral
epitopes more closely than standard vaccines (live attenuated
and killed vaccines are often altered in their protein structure
and antigenicity)
 Plasmid vectors can be constructed and produced quickly and
the coding sequence can be manipulated in many ways
 DNA vaccines encoding several antigens or proteins can be
delivered to the host in a single dose

52.  They are temperature stable making storage and transport much
easier.
 Rapid and large-scale production are available at lower costs
than traditional vaccines
 They have better therapeutic potential for ongoing chronic viral
infections

53. DNA vaccines in development :
o Powdermed - has an Influenza vaccine, a hepatitis B vaccine, a
genital herpes vaccine and a genital warts vaccine in clinical
trials
o GlaxoSmithKline - has a DNA vaccine for cancer, HIV and one
multipurpose vaccine for viruses in early clinical trials

54. •Leukaemia (Kings College London)
•Alzhiemer’s disease (Tokyo Metropolitan Institute for
Neuroscience)
•TB (Pohang University of Science and Technology in
South Korea)
•Ebola (Vaccine Research Center ,Washington DC)
•Multiple Sclerosis (Technion-Israel Institute of Technology)
•Malaria (Malaria Programme at the US Naval Medical Research
Inst)

55. References
1. Textbook of Microbiology -Ananthanarayan and Panniker,9th
edition
2. Diagnostic Microbiology –Bailey and Scott, 13th edition
3. Principles and Practice of Infectious Diseases-Mandell,
Doughlas and Bennet, 7th edition
4. Medical Microbiology-Jawetz, Melnick and Adelberg, 26th
edition
5. Internet

56. THANK YOU