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 The first demonstration of bacterial transformation was done with Streptococcus pneumoniae and led to the discovery that DNA is the substance of the genes. The path leading to this epoch-making discovery began in 1928 with the work of an English bacteriologist, Fred Griffith.

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

S injected into mice -> pneumonia -> death 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 Killed S + Live R 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 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.

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 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).