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Bacteria Transformation!

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Title: Bacteria Transformation!


1
Bacteria Transformation!
  • Picking up stray DNA just in case

2
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

3
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

4
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

5
Griffiths Mysterious Transformation Experiment
6
Frederick 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)

7
The 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)

8
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 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.

10
We dont know why but..
  • Some bacteria have specialized membrane proteins
    to bring DNA into their cells
  • Ca stimulates uptake of DNA into bacteria

11
Another interesting thing about bacteria

Aside from their Circular chromosomal DNA,
bacterial have smaller pieces of circular DNA
called PLASMIDS
12
Plasmids
  •     Extrachromosomal DNA in a bacterial cell
    which can replicate independently but which
    cannot integrate into the host chromosome.

13
Drug resistance Plasmids
  •          Drug resistance plasmids are not
    essential for the cell's growth, but confer
    antibiotic resistance.

14
Plasmids 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

17
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18
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

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.

22
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. 

23
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.  

24
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). 

25
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.

26
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). 

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