TRANSFORMATION

1. TRANSFORMATION BY: FAIEJA CHOWDHURY

2. TABLE OF CONTENTS • GLOSSARY • TRANSFORMATION • HISTORY • NATURAL TRANSFORMATION • GENETICS OF TRANSFORMATION • ARTIFICIAL TRANSFORMATION • IMPORTANCE OF TRANSFORMATION • BIBLIOGRAPHY

3. GLOSSARY • COMPETENCE FACTOR-regulate the entire process • NATURAL COMPETENCE- occurs in natural transformation, DNA is taken from an exogenous cell naturally • ARTIFICAL COMPETENCE-occurs in artificial transformation, DNA is taken and incorporated by genetic engineering • EXONUCLEASE-a nuclease that releases one nucleotide at a time (serially) beginning at one of a nucleic acid  • ENDONUCLEASE-a nuclease that cleaves nucleic acids at interior bonds and so produces fragments of various sizes  • DSDNA-double-stranded-DNA • SSDNA-single-stranded-DNA

4. TRANSFORMATION • Process by which a host cell, mainly bacteria, takes exogenous DNA • Host organism takes in foreign DNA and expresses the foreign gene. • Either a natural process--that is, one that has evolved in certain bacteria--or it may be an artificial process whereby the recipient cells are forced to take up DNA by a physical, chemical, or enzymatic treatment • Exogenous DNA (DNA that is outside the host cell), is taken into a recipient cell where it is incorporated into the recipient genome, changing the genetic makeup of the bacterium

5. HISTORY • In 1928 when he performed experiments with Diplococcus pneumonia -- bacteria that causes pneumonia. • Two forms: S-strain form: has a smooth polysaccharide capsule and R-strain: lacks polysaccharide capsule, gives it a rough appearance. • Mice were injected with S- strain bacteria: died • Injected with R-strain: lived • With heat-killed S- strain bacteria: lived. • R-strain bacteria & with the heat-killed S-strain: died. • Oswald Avery, Colin McCleod, and Maclyn McCarty continued Griffith’s research, using biochemical testing, found out that only DNA could cause the transformation.

6. NATURAL TRANSFORMATION • Physiological process genetically encoded in bacteria. • Bacteria become "competent" for taking up exogenous DNA. • Bacterial culture reaches adequate density • Concentration of competence factor reaches optimum • Bacteria are now capable to bind receptors on the outside of the cell. • An internal signal then turn on the gene expression needed for the transformation • Thus, competence development is controlled by cell density • Competence complex is exposed by autolysin( increases the cell permeability) • Double-stranded DNA are limited to a specific receptor located on the surface of competent cells • Bound fragments are digested by endonuclease(fragements~15Kbp)

8. CONTINUED…. • DNA UPTAKE • -One strand is degraded by the exonuclease • -Second strand enter the cell • Recombination enzymes of recipient cell bind the single-strand DNA • Align it with its corresponding DNA on the recipient chromosome • Recombines the new DNA into the chromosomes • Incorporates genetic differences that exist when DNA is entered. • Cell enters Eclipse phase -time required to covert ssDNA into a stable dsDNA form.

10. GENETICS OF TRANSFORMATION • Requires expression of the late competence genes whose products mediate DNA binding and uptake. • ComC - cuts ComG allowing it to be no longer an integral membrane protein • ComG - permits the access of DNA to ComEA • ComEA - receptor that binds DNA for import • ComEC - forms aqueous transport pathway which DNA enters the cell • ComFA- helicase that functions with ComEA and ComEC so that the ssDNA enters the cell

11. 1 COMPLEX!!

12. CONTINUED… • ComK is a transcription activator • 1998: Leendert W. Hamoen & colleagues: ComK recognizes short A/T-rich sequences arranged in a unique, flexible pattern along the DNA helix. • Showed first that ComK is sufficient to activate transcription at comG promoter. • determined that ComK did not bend DNA when it binds to it - suggesting that a specific interaction is required. • Using gel mobility shift , they showed that four molecules of ComK bind at each promoter. • Hydroxyl-radical foot printing analysis of ComK provided a more detailed look at the binding region and allowed them to conclude that ComK binds to an AT rich sequence AAAAN5TTTT

13. ARTIFICAL TRANSFORMATION • Because most species cannot take DNA from an outside source, it can be done by chemical, physical or enzymatic treatment.  • Plasmids, small circular pieces of DNA, are vectors. • Genetic engineers use artificial transformation by introducing genetically altered sequences into recipient cells.  • First method: where cells are shocked and they use calcium chloride to make the cell more permeable. The DNA is then inserted. • Can also use rubium and magnesium for permeability of cell membrane • Second method - electroporation • Use calcium chloride to allow permeability of cell membrane

14. CONTINUED… • Electroporation: • short bursts of current are passed through a solution containing bacteria at high voltage • current makes the cell membrane leaky (porous) for a short time, allowing the cells to take up DNA molecules from the solution. • By closing the right hand switch, the capacitor is charged • By closing the left hand switch, the direct current is discharged - this disrupts the membrane and uses gel electrophoresis for the DNA to the cell

15. IMPORTANCE OF TRANSFORMATION • Expression of medically useful recombinant proteins such as insulin for treating a disease • Vaccines for prevention of disease • Expression of proteins that give bacteria the ability to survive in particular environments such as to "clean up" contaminated environments

16. BIBLIOGRAPHY • http://www.bookrags.com/research/transformation-gen-04/ • http://biochem4.okstate.edu/~biocukm/MG/MGW4/MG431.html • http://www.sciencebuddies.org/mentoring/project_ideas/BioChem_p013.shtml • http://www.mnstate.edu/provost/transformationProtocol.pdf

17. THE END ! EEEEWWWWWW!!!