Transcript Bacteria Transformation! - Richmond School District

Bacteria Transformation!
Picking up stray DNA just in
case…
History
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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
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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
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S = Virulent
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coated with a
polysaccharide
which makes it
infective, smooth
(S) appearance
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R = Avirulent
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lacking capsules
rough (R) colonies
harmless
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Griffith’s Mysterious
Transformation Experiment
Frederick Griffiths studied the R & S strains by injecting
them into mice
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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:
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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 R
No Disease
Live S in
Blood?
No
Live S
Death
Yes
Killed S
No Disease
No
Killed S + Live Death
R
Yes
The Transforming Factor was Found to be DNA
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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 don’t know why but..
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Some bacteria have
specialized
membrane proteins to
bring DNA into their
cells
Ca stimulates uptake
of DNA into bacteria
Another interesting thing about
bacteria:
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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.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Drug resistance Plasmids
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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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
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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)
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Plasmids
Allow
Bacteria to
Share Genes
Rapidly
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Our Experiment:
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We have
been GIVEN
some
“competent E
coli” (bacteria
that have
been treated
with CaCl2)
They are
frozen and
not very
“happy” =
fragile
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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
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Then we heat
shock the
Ecoli/plasmid
mixture which
helps the
plasmids get
IN to the
Ecoli
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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?
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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?
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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?
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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?
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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).