Title: Bacteria Transformation!
1Bacteria Transformation!
- Picking up stray DNA just in case
2History
- 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
3Evidence 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
4Variants of Streptococcus pneumoniae
- S Virulent
- coated with a polysaccharide which makes it
infective, smooth (S) appearance - R Avirulent
- lacking capsules rough (R) colonies
- harmless
5Griffiths Mysterious Transformation Experiment
6Frederick Griffiths studied the R S strains by
injecting them into mice
- S injected into mice -gt pneumonia -gt death
- R injected into mice -gt harmless
- Also, boiled S injected into mice -gt harmless
(bacteria killed by boiling)
7The Griffiths did a strange experiment and got a
strange result
- Boiled S live R injected into mice -gt pneumonia
-gt 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)
8Summary 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 R Death Yes
The Transforming Factor was Found to be DNA
9- 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.
10We dont know why but..
- Some bacteria have specialized membrane proteins
to bring DNA into their cells - Ca stimulates uptake of DNA into bacteria
11Another interesting thing about bacteria
Aside from their Circular chromosomal DNA,
bacterial have smaller pieces of circular DNA
called PLASMIDS
12Plasmids
- Extrachromosomal DNA in a bacterial cell
which can replicate independently but which
cannot integrate into the host chromosome.
13Drug resistance Plasmids
- Drug resistance plasmids are not
essential for the cell's growth, but confer
antibiotic resistance.
14Plasmids are like viruses, but have no
extra-cellular phase
HIV
15- 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)
16- Plasmids Allow Bacteria to Share Genes Rapidly
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18Our Experiment
- We have been GIVEN some competent E coli
(bacteria that have been treated with CaCl2) - They are frozen and not very happy fragile
19- 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
20- Then we heat shock the Ecoli/plasmid mixture
which helps the plasmids get IN to the Ecoli
21- 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.
22What 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.
23How 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.
24Why 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).
25How 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.
26What 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).
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