TRANSFORMATION

BY:

FAIEJA CHOWDHURY

TABLE OF CONTENTS

GLOSSARYTRANSFORMATIONHISTORYNATURAL TRANSFORMATIONGENETICS OF TRANSFORMATIONARTIFICIAL TRANSFORMATIONIMPORTANCE OF TRANSFORMATIONBIBLIOGRAPHY

GLOSSARY

1) COMPETENCE FACTOR-regulate the entire process2) NATURAL COMPETENCE- occurs in natural

transformation, DNA is taken from an exogenous cell naturally

3) ARTIFICAL COMPETENCE-occurs in artificial transformation, DNA is taken and incorporated by genetic engineering

4) EXONUCLEASE-a nuclease that releases one nucleotide at a time (serially) beginning at one of a nucleic acid 

5) ENDONUCLEASE-a nuclease that cleaves nucleic acids at interior bonds and so produces fragments of various sizes 

6) DSDNA-double-stranded-DNA7) SSDNA-single-stranded-DNA

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

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: livedWith 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.

NATURAL TRANSFORMATIONPhysiological 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)

CONTINUED….

DNA UPTAKE -One strand is degraded by the

exonuclease-Second strand enter the cell

Recombination enzymes of recipient cell bind the single-strand DNAAlign it with its corresponding DNA on the recipient chromosomeRecombines the new DNA into the chromosomesIncorporates genetic differences that exist when DNA is entered.Cell enters Eclipse phase -time required to covert ssDNA into a stable dsDNA form.

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 proteinComG - permits the access of DNA to ComEAComEA - receptor that binds DNA for importComEC - forms aqueous transport pathway which DNA enters the cellComFA- helicase that functions with ComEA and ComEC so that the ssDNA enters the cell

1

COMPLEX!!

ComK is a transcription activator1998: 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

CONTINUED…

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

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

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

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

THE END !

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