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TRANSFORMATION
BY:
FAIEJA CHOWDHURY
TABLE OF CONTENTS
GLOSSARY
TRANSFORMATION
HISTORY
NATURAL TRANSFORMATION
GENETICS OF TRANSFORMATION
ARTIFICIAL TRANSFORMATION
IMPORTANCE OF TRANSFORMATION
BIBLIOGRAPHY
GLOSSARY
1) COMPETENCE FACTOR-regulate the entire process
2) 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-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: 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.
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)
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.
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
1
COMPLEX!!
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
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
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/transform
ation-gen-04/
http://biochem4.okstate.edu/~biocukm/MG/M
GW4/MG431.html
http://www.sciencebuddies.org/mentoring/pr
oject_ideas/BioChem_p013.shtml
http://www.mnstate.edu/provost/transformati
onProtocol.pdf
THE END !
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