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Bacterial

Transformation and Plasmid Purification

Chapter 5: Background

Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com 2

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


Origin of Plasmids

Joshua Lederberg and William Hayes

independently discovered plasmids

while studying conjugation – 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


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


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)


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)


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


Transcriptional Regulation of Plasmids

How operons work

– Jacob and Monod in 1961

discovered how the lac

operon work in bacteria



How pBAD operon works

– An operon in which

arabinose is the inducer

instead of lactose

• Different operons have

different inducers



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


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


Transforming Cells

Two major methods of transformation

– Calcium chloride

– Electroporation


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


Transformation of Bacteria

Play video: Bacterial Transformation


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


Why Calcium Chloride?

Helps to neutralize the

charge on DNA

molecule, increasing

probability of that

molecule moving into

the cell

Ca++

Ca++

O CH2

O

P O

O

O Base

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

O Ca++


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


Electroporation

Electroporation works by

– Using electricity to disrupt the bacterial

wall and membranes

– Plasmids move in during disruption


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


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


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 X-

gal and will be white in color

– Antibiotic selection is also

used to ensure that the

bacteria were successfully

transformed initially


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


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)


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



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



Steps:

– Next, calculate transformation efficiency by dividing the CFU

by the amount of DNA spread on plate


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


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)


Purification of Plasmids


Purifying Plasmid

Play video: Alkaline Lysis Miniprep


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


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 double-

stranded 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


DNA Quantitation


Determining the Concentration of DNA

Play video: DNA Quantitation Using a

Spectrophotometer


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


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


Chapter 5 Summary

Background

• History of Plasmids

• Plasmid Structure and Function

Uses of Plasmids

• Recombinant Plasmids

• Transcriptional Regulation

• Transformation

Transformation

• Selection

• Efficiency

• Purification of Plasmids

DNA Quantitation

• Spectrophotometer

• Purity

• Fluorometer