Presentation on theme: "Bacteria Transformation!"— Presentation transcript:
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 cellsThis was first discovered in 1928 by Frederick Griffith
3Evidence for DNA as the genetice material Transformation1928Frederick Griffith laid the foundation of the identification of DNA as the genetic material with his experiment on transformation in the bacterium Streptococcus pneumoniaeThe 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.
4Variants of Streptococcus pneumoniae S = Virulentcoated with a polysaccharide which makes it infective, smooth (S) appearanceR = Avirulentlacking capsules rough (R) coloniesharmless
6S injected into mice -> pneumonia -> death Frederick Griffiths studied the R & S strains by injecting them into miceS injected into mice -> pneumonia -> deathR injected into mice -> harmlessAlso, boiled S injected into mice -> harmless (bacteria killed by boiling)
7The Griffiths did a strange experiment and got a strange result: Boiled S + live R injected into mice -> pneumonia -> deathThis was not expected because boiled S and live R were harmless by themselvesTook blood samples and found live S in the dead miceConcluded 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 InjectedResultLive S in Blood?Live RNo DiseaseNoLive SDeathYesKilled SKilled S + Live RThe Transforming Factor was Found to be DNA
9Today, 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 don’t know why but..Some bacteria have specialized membrane proteins to bring DNA into their cellsCa 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
15Plasmids used for molecular cloning have been artificially created by recombining fragments of various existing plasmids.Examples of PlasmidspBR322pUC19 (the one we will use)
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
19We 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
20Then we heat shock the Ecoli/plasmid mixture which helps the plasmids get IN to the Ecoli
21Now that the plasmids are inside, we add some SOC medium SOC media feeds the Ecoli and allows it to grow/divideThe plasmids will also divide inside the EcoliIf 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 TRANSDUCTION3) 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).