Now, what is a plasmid ?
Let us restart with our current Understanding of Plasmids
Plasmid is autonomously replicating, extrachromosomal circular DNA molecules,
distinct from the normal chromosomal DNAs and nonessential for cell survival under
nonselective conditions.
Episome no longer in use.
They usually occur in bacteria, sometimes in eukaryotic organisms (e.g., the 2-um-ring in
yeast S. cerevisiae).
Sizes: 1 to over 400 kb.
Copy numbers: 1 - hundreds in a single cell, or even thousands of copies.
Every plasmid contains at least one DNA sequence that serves as an origin of replication
or ori (a starting point for DNA replication, independently from the chromosomal DNA).
Schematic drawing of a bacterium with its
plasmids.
(1) Chromosomal DNA. (2) Plasmids
Types of Bacterial Plasmids
Based on their function, there are five main classes:
Fertility-(F)plasmids: they are capable of conjugation or mating.
Resistance-(R) plasmids: containing antibiotic or drug resistant gene(s). Also known as R-factors, before
the nature of plasmids was understood.
Col-plasmids: contain genes that code for colicines, proteins that can kill
other bacteria.
Degrative plasmids: enable digestion of unusual substances, e.g., toluene or
salicylic acid.
Virulence plasmids: turn the bacterium into a pathogen.
Plasmids can belong to more than one of these functional groups.
Amp-R
Antibiotic resistance
ori
Kan-R
Schematic drawing of a plasmid with antibiotic resistances
R-plasmids often contain genes that confer a selective advantage to the
bacterium hosts, e.g., the ability to make the bacterium antibiotic resistant.
Some common antibiotic genes in plasmids: ampr, APH3’-II (kanamycin),
tetR (tetracycline),catR (Chloramphenicol), specr (spectinomycin or
streptomycin), hygr (hygromycin).
Some antibiotics inhibit cell wall synthesis and others bind to ribosomes to
inhibit protein synthesis
Development of Plasmid Vectors
Plasmids serve as important tools in genetics and biochemistry labs, where they are
commonly used to multiply or express particular genes.
Plasmids used in genetic engineering are called vectors.
Vectors are vehicles to transfer genes from one organism to another and typically
contain a genetic marker conferring a phenotype.
Most also contain a polylinker or multiple cloning site (MCS), with several commonly
used restriction sites allowing easy insertion of DNA fragments at this location.
Many plasmid vectors are commercially available.
Old vector pBR322: 4.36kb, Ampicilin-R, Tetracylin-R, 15-20 copies/cell
Old vectors pUC18/19: 2.69kb, Ampicilin-R, LacZ operon, 500-700 copies
Stratagen pBS-KS: 3.0kb, Ampicilin-R, LacZ operon, 500-700 copies/cell
Promega pGEM-T: 3.0 kb, Ampicilin-R, LacZ operon, 500-700 copies/cell
Invitrogen TOPO-TA: 3.96kb, Ampicilin-R, Kan-R, LacZ, 500-700 copies
pCAMBIA vectors: >10kb, Amp-R/Kan-R/Hyg-R, LacZ, 1-3 copies
Plasmid Vectors
MCS
Application of Plasmid Vectors
In Molecular Cloning
How it works?
(a) Initially, the gene to be
replicated is inserted in a
plasmid or vector.
(b) The plasmids are next inserted
into bacteria by a process called
transformation.
(c) Bacteria are then grown on
specific antibiotic(s).
(d) As a result, only the bacteria with
antibiotic resistance can survive
and will be replicated.
Application of Plasmid Vectors
In Pharmaceutical and Agriculture Bioengineering
One of the major uses of plasmids is to make large amounts of proteins.
In this case, bacteria or other types of host cells can be induced to produce large
amounts of proteins from the plasmid with inserted gene, just as the bacteria
produces proteins to confer antibiotic resistance. This is a cheap and easy way of
mass-producing a gene or the protein — for example, insulin, antibiotics, antobodies
and vaccines.
Green Algae for
antibody
production
Transgenic
Arabidopsis
expressing
GFP to study
PDI
functions
Plasmid Isolation from Bacteria
How to rapidly isolate plasmid?
(a) Inoculation and harvesting the bacteria
(b) lysis of the bacteria (heat, detergents (SDS or
Triton-114), alkaline(NaOH)),
(c) neutralization of cell lysate and separation of
cell debris (by centrifugation),
Or other
cell types
Plasmid DNA Isolation continued
Tranditional
Ways
Midi Prep
Mini Prep
(d) collecting plasmid
DNA by centrifugation
(after ethanol precipitation
or through filters positively charged silicon
beads),
(e) check plasmid DNA
yield and quality (using
spectrophotometer and gel
electrophoresis).
spectrophotometer and gel electrophoresis
1. Isolation of plasmid DNA
 Plasmid DNA can be isolated from bacterial cells
using alkaline/SDS based mini-plasmid
purification method.
 The method of isolating plasmid DNA using
alkaline lysis with SDS was first reported by
Birnboim HC and Doly J. in 1979 (Nucleic Acids
Res. 1979 Nov 24;7(6):1513-23). “A rapid alkaline
extraction procedure for screening recombinant
plasmid DNA”.
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1. Isolation of plasmid DNA
 This method has been used for more than 25 years
to isolate plasmid DNA from E. coli.
 It is very simple and permits the analysis of 100 or
more clones per day by gel electrophoresis.
 The plasmid DNA is pure enough to be digestible
by restriction enzymes.
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Isolation of plasmid DNA
 About 10 years ago, this method
was modified so that very pure
plasmid DNA can be purified
using a column specially packed
with glass fiber.
 The plasmid DNA purified
through those columns can be
used directly for PCR, cloning,
sequencing, in vitro
transcription, synthesize of
labelled hybridization probes,
microinjection, electroporation
and transfection, etc.
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CH3
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Bacterial
chromosome
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Plasmid
CH2
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previous
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thetoanimation
replay the animation.
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CH2
 SDS is a strong ionic detergent with a
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o
CH2
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O
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S
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O
o
Sodium dodecyl sulfate
(SDS)
negative charge.
 At high pH, SDS can open the bacterial
cell wall, denature chromosomal DNA
and proteins, and release plasmid DNA
into the supernatant.
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Click on animation to start playing
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3’
• SDS/alkaline solution
can denature
chromosomal DNA by
disrupting the hydrogen
bonds between the two
complementary strands
of nucleotides.
• However, this treatment
cannot destroy the
covalently closed circular
plasmid DNA because
they are topologically
intertwined.
5’
G
T
T
A
T
A
G
C
G
C
T
A
C
G
A
5’
A
A
T
Circular dsDNA
C
T
3’
Hydrogen-bonded
base pairs
dsDNA in
supercircular form
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