Transcript lect 8- Transformation

Methods of
Transformation/Transfection
Introduction
• Transformation
– the exchange of genetic material between
strains of bacteria by the transfer of a
fragment of naked DNA from a donor cell
to a recipient cell
Natural Transformation
• Some bacteria are naturally competent.
• These bacteria have special protein
spanning on their envelope
• e.g Bacillus subtilis, Haemophilus influenzae
• Regulate transformation competence by
quorum sensing
Quorum Sensing
• Competent bacteria take in DNA from
environment, when the concentration of
cells in the environment is high this is called
quorum sensing.
Process of Natural
Transformation
Process of Natural
Transformation
Introduction
• Transfection
– Infection of a cell with viral nucleic acid,
resulting in subsequent replication of the
virus in the cell.
Terms in Transformation
Methods
• Transient
–DNA is degraded and transgene
expression is lost
• Stable Transfection/Transformtaion
–DNA is introduced in such a way that it is
maintained indefinitely
Transformation of Bacteria
• Methods:
– Electroporation
– Freeze Thaw Method
– Calcium Chloride Mediated
Transformation
– Calcium Phosphate Mediated
Transformation
Calcium Chloride Method
• Treatment with calcium
chloride in the early log
phase of growth for
Competence
• Bacterial cell membrane is
permeable to chloride ions,
but is non-permeable to
calcium ions
Chloride
Ions
E.Coli
Calcium Chloride Method
• As the chloride ions enter
the cell, water molecules
accompany the charged
particle
• Influx of water causes the
cells to swell and is
necessary for the uptake
of DNA
• The exact mechanism of
this uptake is unknown.
Calcium Chloride Method
• DNA of interest is then added to the
cells
Calcium Chloride Method
• Calcium chloride
treatment be followed
by addition of DNA of
interest then by heat.
• The heat shock step is
necessary for the
uptake of DNA.
Calcium Chloride Method
• Temperatures > 42degC: Bacteria’s
ability to uptake DNA reduces
• Extreme temperatures: Bacteria dies.
Calcium Chloride Method
• After the heat shock step intact plasmid
DNA molecules replicate in bacterial
host cells
• To help the bacterial cells recover from
the heat shock cells are briefly
incubated with non-selective growth
media
Calcium Chloride Method
• As the cells recover,
plasmid genes are
expressed
• Bacterial colonies
selected using antibiotic
selection techniques
Freeze Thaw Method
Making cells competent:
•
•
•
•
Cells are grown to log phase
Cells are centrifuged at 5000rpm at room
temp.
Pellet is washed.
Cells are then froze in liquid nitrogen and
stored at -70 degree centigrade.
Freeze Thaw Method
•
Transformation and Recovery :
1. Competent cells are thawed on ice and DNA
is added.
2. Cells are then incubated for recovery
3. Transformed cells are selected by antibiotic
selection
Electroporation
• Electroporation is a method of transformation
that allows the introduction of foreign DNA into
host cells (prokaryotic or eukaryotic) via the
application of high-voltage electric pulses
Electroporation
• Cells are placed in buffer and put in to
electroporator.
• DNA is added and subjected to a high-voltage
electrical pulse of defined magnitude and length.
• The cells are then allowed to recover.
• Selected by antiobiotic selection technique.
Electroporation
• It is the most efficient method.
• 2.5 kV,200 ohms about 5 milli seconds
required for bacterial cell.
Any More Questions?
Thank You