Bacterial
Transformation
and Plasmid Purification
Chapter 5: Background
History of Transformation and Plasmids
 Bacterial methods of DNA transfer
– Transformation: when bacteria take up DNA from their
environment
– Conjugation: process of transferring DNA by a pilus
(bridge) from one bacteria to another
– Transduction: when bacterial DNA is transferred from
one bacteria to another by viruses
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Origin of Plasmids

Joshua Lederberg and William Hayes
independently discovered plasmids
while studying conjugation
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1952 Lederberg proposed the name plasmid
1961 Tsutomu Watanabe and Toshio
Fukasawa found that some plasmids carried
antibiotic resistance genes
1962 Allan Campbell determined that plasmids
were circular
1973 Peter Lobban proposed using restriction
enzymes to help recombine DNA
1973 Stanley Cohen, Annie Chang, Herbert
Boyer, and Robert Helling published a paper
describing how to construct a functional
plasmid
1976 Herbert Boyer and Robert Swanson
founded Genentech using plasmids to
manufacture insulin
2009 Genentech was sold to Roche for $46
billion
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Plasmids: Structure and Function

Most are extrachromosomal loops of
DNA that can self-replicate in the
cytosol of bacteria
– They have an origin of replication (ori
on the map)
– Are designated with a “p” in the name
– Have genes that code for proteins.
They are symbolized by an arrow in the
direction of transcription
• Genes are preceded by a promoter
– The location for RNA polymerase to
bind
• They are followed by a terminator
– The location that causes the
polymerase to stop transcribing
– Number of plasmids ranges from 5 to
1,000 per bacterial cell
• Low copy number plasmids
• High copy number plasmids
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Plasmid Uses
 Two main uses
– To express recombinant
proteins
– To house genes that have
been cloned
• These can then be
placed into other
organisms (e.g. corn)
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Modern Plasmids
 Plasmids are constructed to make cloning easy.
They have an area called a multiple cloning site
(MCS) that has a series of unique restriction enzyme
recognition sites
– This MCS is used to open up the plasmid to receive the
gene of interest
Plasmid with a gene
(red) inserted into
the MCS (green)
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Recombinant DNA Using Plasmids
 Steps
– Extract and purify plasmid
and DNA of interest
– Digest plasmid and DNA
of interest with restriction
enzymes
• PCR can be used to
amplify gene of interest
– Mix the two different DNA
fragments together and
add DNA ligase
– Transform plasmid into
host cell
– Grow and select for cells
that have insert
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Transcriptional Regulation of Plasmids
 How operons work
– Jacob and Monod in 1961
discovered how the lac
operon work in bacteria
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Transcriptional Regulation of Plasmids
 How pBAD operon works
– An operon in which
arabinose is the inducer
instead of lactose
• Different operons have
different inducers
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Transcriptional Regulation of Plasmids
 If the three genes BAD are
cut out by restriction
enzymes and GFP is
ligated in their place, a
recombinant operon is
produced that expresses
GFP
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Other Types of Plasmids
 Shuttle plasmids
– Plasmids that can be inserted into bacteria initially to be
cloned, then transformed into eukaryotic cells once
duplicated and isolated
• For example, to grow in E. coli, a plasmid needs a prokaryotic
origin of replication and an antibiotic-resistant gene
• To grow in a eukaryote, it would need a eukaryotic origin of
replication, a sequence for a poly A tail, a promoter, and a
terminator sequence that would function in a eukaryotic cell
 Ti plasmid
– Found naturally in Agrobacterium tumefaciens
– Causes crown gall disease in plants
– Can be modified to carry genes of interest into plants
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Transforming Cells
 Two major methods of transformation
– Calcium chloride
– Electroporation
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Calcium Chloride Transformation Steps
 Suspend bacterial colonies in 50 mM (0.05 M)
calcium chloride
 Add plasmid DNA
 Place tubes on ice
 Heat shock at 42ºC and place on ice
 Incubate with nutrient broth
 Streak plates
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Transformation of Bacteria
 Play video: Bacterial Transformation
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How the Calcium Chloride Method Works
 In the presence of
calcium chloride,
plasmids are mixed
with bacteria and
heat shocked
 Plasmids move into
the bacteria
GFP
Beta-lactamase
Ampicillin
Resistance
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Why Calcium Chloride?
 Helps to neutralize the
charge on DNA
molecule, increasing
probability of that
molecule moving into
the cell
Ca++
Ca++
O
O P O
O
CH2
Base
O
Sugar
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
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What Happens in Each Step?
 Incubate on ice
– Slows fluid cell membrane
 Heat shock
– Increases permeability of membranes
 Nutrient broth incubation
– Allows beta-lactamase expression
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Electroporation
 Electroporation works by
– Using electricity to disrupt the bacterial
wall and membranes
– Plasmids move in during disruption
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Other Methods of Moving DNA into Cells
 Biolistics
– Using microparticles to
shoot or blast small
particles coated with DNA
into cells
• Plants have a cell wall that
is difficult to disrupt to
move DNA into cells
 Transfection
– Plasmids are placed into
lipid vesicles
• The vesicles merge with
cell membranes and
deliver DNA into the cells
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Methods to Select Transformed Cells
 Antibiotic selection
– When bacteria are plated onto agar that contains antibiotic
– Bacteria that successfully incorporate a plasmid can grow in
the presence of antibiotics due to the new enzyme on the
plasmid
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Selection of Transformed Cells
 Blue-white screening
– The β-galactosidase
enzyme cleaves X-gal
converting the X-gal into a
blue color
– If a gene is successfully
inserted into the MCS
(shown in green), then it
disrupts the cleavage of Xgal and will be white in color
– Antibiotic selection is also
used to ensure that the
bacteria were successfully
transformed initially
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Selection of Transformed Cells
with an Insert
 pJET1.2 plasmid
– The plasmid contains the
Eco47IR gene, which
codes for a restriction
enzyme that is toxic to
E. coli
– If an insert is successfully
inserted, then the
Eco47IR gene is
disrupted and the bacteria
survive
– Antibiotic selection is still
part of the plasmid
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Transformation Efficiency
 Measurement of the number of transformed cells
per microgram of plasmid DNA utilized
– Electroporation is the most efficient method
– Transformation with plasmid DNA is more efficient than
with plasmid that has been ligated
– Transformation with ligated DNA requires cells with very
high transformation efficiency (>106 CFU/µg of DNA)
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Calculating Transformation Efficiency
 Example:
– 50 ng of plasmid DNA is transformed into a final
transformation volume of 500 μl, and 10 μl of this volume is
spread on an agar plate. Assume that 60 CFU are observed
on the agar plate
• Note: 1 μg is 1,000 ng, so 50 ng = 0.05 μg of DNA
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Calculating Transformation Efficiency
 Steps:
– First, count the number of colonies growing on the
LB/ampicillin (LB/amp) agar plate. In this case, the CFU is
60
– Next, determine the amount of plasmid DNA (in μg) spread
on the LB/amp agar plate. In this example, only 10 μl of a
500 μl transformation was spread on the plate
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Calculating Transformation Efficiency
 Steps:
– Next, calculate transformation efficiency by dividing the CFU
by the amount of DNA spread on plate
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Maximizing Transformation Efficiency
 E. coli divides once
every 17 minutes
– Cells for purification of
plasmids are typically
harvested late in growth
phase E. coli is optimally
grown for 16–24 hours at
37ºC with shaking
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Purification of Plasmids
 Alkaline lysis method
– Uses detergent to lyse cells, releasing the DNA into solution
– Alkaline environment makes DNA single-stranded (plasmid
and genomic)
– Acid allows the smaller plasmids to re-anneal; the longer
genomic DNA strands only partially re-anneal
– Centrifuging pulls cell debris and genomic DNA to the
bottom of the cell
– Plasmids are in the supernatant (liquid on top)
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Purification of Plasmids
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Purifying Plasmid
 Play video: Alkaline Lysis Miniprep
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DNA Quantitation
 Gel quantitation
– Matching the intensity
of bands on a gel with a
band on the same gel
that has a known
quantity
Unknown
DNA band to
quantify
Known
bands to
compare
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DNA Quantitation
 Spectrophotometric quantitation
– DNA absorbs UV light at 260 nm
– An absorbance of 1 at 260 (A260) is equivalent to 50 µg/ml of doublestranded DNA
• So an absorbance of 0.5 would be equivalent to 25 µg/ml
• Single-stranded DNA with an absorbance of 1 is 33 µg/ml
• Single-stranded RNA with an absorbance of 1 is 40 µg/ml
– Often DNA is diluted before it is quantified, because it is very
precious and one would not want to use it up to quantify it. It is often
diluted from tenfold to 100-fold
– If the DNA is diluted,
the dilution must be
accounted for in the
final concentration
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DNA Quantitation
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Determining the Concentration of DNA
 Play video: DNA Quantitation Using a
Spectrophotometer
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DNA Purity
 Spectrophotometer can be used to test DNA purity
– Often DNA is contaminated with protein. Proteins absorb
UV at 280 nm
– This is tested by taking the absorbance at 260 nm and
280 nm
• A260:A280
• Pure DNA is >1.8
• Pure RNA is >2.0
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DNA Quantitation
 Fluorometer
– DNA is bound to a dye that
fluoresces at a particular
wavelength
– The fluorometer excites the
sample at a particular wavelength
and then measures emitted
wavelengths
• Can measure samples at a much
lower concentration than a
spectrophotometer
– >1µg for a spectrophotometer
– nanograms for a fluorometer
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Chapter 5 Summary
Background
Uses of
Plasmids
Transformation
DNA
Quantitation
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• History of Plasmids
• Plasmid Structure and Function
• Recombinant Plasmids
• Transcriptional Regulation
• Transformation
• Selection
• Efficiency
• Purification of Plasmids
• Spectrophotometer
• Purity
• Fluorometer
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