Bacterial

Transformation and Plasmid Purification

Chapter 5: Background

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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 – 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 CH 2

O

P O

O

O Base

CH 2

O

P

O

O

O

Base

OH

Sugar

Sugar

O Ca ++

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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 X-

gal 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