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 : Competent cells are thawed on ice and DNA is added. Cells are then incubated for recovery 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.

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