BacteriaTransformation ppt

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

http://en.wikipedia.org/wiki/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

Griffith’s 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 R

Death 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.

http://en.wikipedia.org/wiki/DNA

We don’t 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.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Drug resistance Plasmids

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

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







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?How can DNA be put into bacteria?

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

2) Viruses can insert DNA into bacteria by 2) Viruses can insert DNA into bacteria by TRANSDUCTION TRANSDUCTION

3) 3) can insert DNA into bacteria using chemicals or can insert DNA into bacteria using chemicals or electricity, which is called TRANSFORMATION. electricity, which is called TRANSFORMATION.

During this lab, we will 'poke holes' in the bacteria During this lab, we will 'poke holes' in the bacteria using chemicals, allowing the DNA to flow into the using chemicals, allowing the DNA to flow into the bacteria- this is called BACTERIAL bacteria- this is called BACTERIAL TRANSFORMATION. 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).

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BacteriaTransformation ppt