Bacteria Transformation!

Picking up stray DNA just in

case

History
Bacteria Can Get
Genes from Naked
DNA=
Transformation
Sometimes naked
DNA is taken up by
bacterial cells
This was first
discovered in 1928 by
Frederick Griffith

Evidence for DNA as the

genetice material

Transformation
1928
Frederick Griffith laid the foundation of the
identification of DNA as the genetic material
with his experiment on transformation in the
bacterium Streptococcus pneumoniae

Variants of Streptococcus pneumoniae

S = Virulent

coated with a
polysaccharide
which makes it
infective, smooth
(S) appearance

R = Avirulent

lacking capsules
rough (R) colonies
harmless

Griffiths Mysterious
Transformation Experiment

Frederick Griffiths studied the R & S strains by injecting

them into mice

S injected into mice -> pneumonia -> death

R injected into mice -> harmless
Also, boiled S injected into mice -> harmless
(bacteria killed by boiling)

The Griffiths did a strange experiment and got a

strange result:

Boiled S + live R injected into mice ->

pneumonia -> death
This was not expected because boiled S and
live R were harmless by themselves
Took blood samples and found live S in the dead
mice
Concluded that some factor, a "transforming
principle", from the dead S had converted some
R bacteria into S bacteria (a genetic change)

Summary of Griffith's experiments

Injected

Result

Live S in
Blood?

Live R

No Disease

No

Live S

Death

Yes

Killed S

No Disease

No

Killed S + Live Death

R

Yes

The Transforming Factor was Found to be DNA

Today, we know that the "transforming principle"

Griffith observed was the DNA of the S strain
bacteria. While the bacteria had been killed, the
DNA had survived the heating process and was
taken up by the R strain bacteria. The S strain
DNA contains the genes that form the protective
polysaccharide capsule. Equipped with this
gene, the former R strain bacteria were now
protected from the host's immune system and
could kill it.

We dont know why but..

Some bacteria have
specialized
membrane proteins to
bring DNA into their
cells
Ca stimulates uptake
of DNA into bacteria

Another interesting thing about

bacteria:

Aside from their Circular

chromosomal DNA,
bacterial have smaller
pieces of circular DNA
called PLASMIDS

Plasmids
Extrachromosomal
DNA in a bacterial cell
which can replicate
independently but
which cannot integrate
into the host
chromosome.
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Drug resistance Plasmids

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Drug resistance
plasmids are not
essential for the cell's
growth, but confer
antibiotic resistance.

Plasmids are like viruses, but

have no extra-cellular phase

HIV

QuickTime and a
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QuickTime and a
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QuickTime and a
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Plasmids used for

molecular cloning have
been artificially created by
recombining fragments of
various existing plasmids.

Examples of Plasmids
pBR322
pUC19 (the one we will
use)

Plasmids
Allow
Bacteria to
Share Genes
Rapidly

QuickTime and a
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QuickTime and a
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QuickTime and a
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Our Experiment:

We have
been GIVEN
some
competent E
coli (bacteria
that have
been treated
with CaCl2)
They are
frozen and
not very
happy =
fragile

We mix the
competent cells
with the plasmid
DNA pUC19.
(they GAVE us
this!)
pUC19 plasmid
has the ampicillin
resistance gene
on it.
Put this mixture
on ice to let the
plasmids stick
to the Ecoli cell
walls

Then we heat
shock the
Ecoli/plasmid
mixture which
helps the
plasmids get
IN to the
Ecoli

Now that the plasmids are inside, we add some

SOC medium
SOC media feeds the Ecoli and allows it to
grow/divide
The plasmids will also divide inside the Ecoli
If the plasmids do reproduce, they will provide the
Ecoli cells with amp resistance so they can grow
on the hostile plates (plates with ampicillin in
them) you will spread the Ecoli on overnight.
Only the transformed cells will be able to grow.

What can we use DNA for?

We can use DNA to code for proteins,

to identify individuals (like when
solving a crime)
do genetic engineering by inserting
foreign DNA into an organism.

How can DNA be put into bacteria?

1) Bacteria can insert DNA into each other by
CONJUGATION (remember the 2 methods of bacteria reprocdution? As
mentioned in class)

2) Viruses can insert DNA into bacteria by

TRANSDUCTION
3) can insert DNA into bacteria using chemicals or
electricity, which is called TRANSFORMATION.
During this lab, we will 'poke holes' in the bacteria
using chemicals, allowing the DNA to flow into the
bacteria- this is called BACTERIAL
TRANSFORMATION.

Why would we want to put DNA into bacteria?

We can use bacteria as little 'factories'

to make more DNA, as they replicate,
or to make protein, by transforming
them with genes for proteins we want
to make (like insulin).

How can we tell DNA is in the bacteria once we

put it there?

The DNA we insert is shaped in a little

circle, called a plasmid.
We can put one, two, or more genes in a
single plasmid.
One of the genes in the plasmid codes
for the ampicillin resistance protein, and
thus will allow bacteria with the plasmid
DNA to grow in the presence of
ampicillin.

What is a plasmid? What is ampicillin?

A plasmid is a small circle of DNA.

Ampicillin is an antibiotic; antibiotics
prevent bacteria from growing.
Ampicillin specifically prevents bacteria
from making cell walls.
ampicillin will not kill bacteria (that already have a cell
wall), but will prevent bacteria from reproducing (because
they can't make new cell walls).