BACTERIAL TRANSFORMATION: BACTERIAL TRANSFORMATION PRESENTATION BY: NISHIKA BHAN M.Sc. M.T. Gene Transfer Mechanisms: Gene Transfer Mechanisms Gene Transfer in Bacteria Vertical Horizontal Vertical Gene Transfer (VGT) When an organism recovers genetic material from its ancestors. Binary fission - asexual reproduction Horizontal Gene Transfer (HGT) Transfer of genes between same species. Any process in which an organism incorporates genetic material from an organism without being the offspring of that organism. PowerPoint Presentation: There are three types of horizontal gene transfer – Transformation Transduction Conjugation TRANSFORMATION: TRANSFORMATION Process by which a cell takes up naked DNA segment from environment, incorporates into its own chromosomal DNA, and finally expresses the trait. Gene transfer resulting from the uptake by a recipient cell of naked DNA from a donor cell. PowerPoint Presentation: Certain bacteria (e.g. Bacillus, Haemophilus , Neisseria , Pneumococcus) can take up DNA from the environment and the DNA that is taken up can be incorporated into the recipient's chromosome. Discovery of Transformation: Discovery of Transformation Griffith’s experiment: Griffith’s experiment Key experiments done by Frederick Griffith in 1928 Griffith used two strains of pneumococcus ( Streptococcus pneumoniae ) bacteria which infect mice – a type S (smooth) and type R (rough) strain. Two strains were distinguished by the appearance of their colonies: Two strains were distinguished by the appearance of their colonies : One strain : VIRULENT , deadly to mice cells of this strain were enclosed in a polysaccharide capsule, giving colonies a smooth appearance ; hence this strain was identified as S TYPE. Other strain : NONVIRULENT , not lethal polysaccharide coat absent, giving colonies a rough appearance ; hence identified as R TYPE. s.pneumoniae: s.pneumoniae Rough colonies (R TYPE) Smooth colonies (S TYPE) Griffith’s Experiment: Griffith’s Experiment PowerPoint Presentation: Griffith found that mice died when they were injected with a mixture of live non capsulated (R, type II) strains and heat killed capsulated (S, type I) strains. Neither of these two when injected alone could kill the mice, only the mixture of two proved fatal. Live S strains with capsule were isolated from the blood of the animal suggesting that some factor from the dead S cells converted the R strains into S type. The factor that transformed the other strain was found to be DNA by Avery, McLeod and McCarty in 1944. PowerPoint Presentation: wikipedia Conclusion: Conclusion While neither alone harmed the mice, the combination was able to kill its host. Griffith concluded that the type R had been "transformed" into the lethal S strain by a "transforming principle" that was somehow part of the dead S strain bacteria. Reason: Reason The S strain covers itself with a polysaccharide capsule that protects it from the host's immune system, resulting in the death of the host, while the R strain doesn't have that protective capsule and is defeated by the host's immune system. Griffith’s Experiment did not prove that DNA was responsible for transformation: Griffith ’ s Experiment did not prove that DNA was responsible for transformation Avery, McCarty, and MacLeod Repeated Griffith’s Experiment: Avery, McCarty, and MacLeod Repeated Griffith ’ s Experiment Oswald Avery Maclyn McCarty Colin MacLeod PowerPoint Presentation: Avery, McCarty, and MacLeod Added the non-deadly Rough Type of Bacteria to the Heat-Killed Smooth Type Carbohydrates Lipids Proteins RNA DNA To the Heat-Killed Smooth Type, added enzymes that destroyed… PowerPoint Presentation: S-Type Carbohydrates Destroyed S-Type Lipids Destroyed S-Type Proteins Destroyed S-Type RNA Destroyed S-Type DNA Destroyed Conclusion: DNA was the transforming factor! Importance in Molecular Biology: Importance in Molecular Biology is a very important basic tool in molecular biology. is used for cloning or to move DNA molecules around between strains. expression of medically useful recombinant proteins such as insulin for treating a disease or vaccines for prevention of disease. Bacterial Transformation: Bacterial Transformation This is a very basic technique that is used on a daily basis in a molecular biological laboratory. purpose of this technique is to introduce a foreign plasmid into a bacteria and to use that bacteria to amplify the plasmid in order to make large quantities of it. Factors affecting transformation : Factors affecting transformation Competence of the recipient :- - some bacteria are able to take up DNA naturally. - take up DNA in a particular time in their growth cycle when they produce a specific protein called a competence factor. - at this stage the bacteria are said to be competent. Other bacteria which do not take up DNA naturally :: Other bacteria which do not take up DNA naturally : Since DNA is a very hydrophilic molecule, it won't normally pass through a bacterial cell's membrane. In order to make bacteria take in the plasmid, they must first be made " competent " to take up DNA. This is done by creating small holes in the bacterial cells by suspending them in a solution with a high concentration of calcium. DNA can then be forced into the cells by incubating the cells and the DNA together on ice, placing them briefly at 42 o C (heat shock), and then putting them back on ice. This causes the bacteria to take in the DNA. Steps in Transformation: Steps in Transformation Step 1: A donor bacterium dies and is degraded.: Step 1 : A donor bacterium dies and is degraded. Step 2: A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium. : Step 2 : A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium. Step 3: The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA. : Step 3 : The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA. 4. Exchange is complete. : http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transformation/transformation.html 4. Exchange is complete. THANK YOU!: THANK YOU!