1. Methods of Transformation/Transfection

2. 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

3. 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

4. Quorum Sensing
Competent bacteria take in DNA from environment, when the concentration of cells in the environment is high this is called quorum sensing.

5. Process of Natural Transformation

6. Process of Natural Transformation

7. Introduction Transfection
Infection of a cell with viral nucleic acid, resulting in subsequent replication of the virus in the cell.

8. 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

9. Transformation of Bacteria
Methods:
Electroporation
Freeze Thaw Method
Calcium Chloride Mediated
Transformation
Calcium Phosphate Mediated Transformation

10. 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

11. 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.

12. Calcium Chloride Method
DNA of interest is then added to the cells

13. 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.

14. Calcium Chloride Method
Temperatures > 42degC: Bacteria’s ability to uptake DNA reduces
Extreme temperatures: Bacteria dies.

15. 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

16. Calcium Chloride Method
As the cells recover, plasmid genes are expressed
Bacterial colonies selected using antibiotic selection techniques

18. 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.

19. 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

20. 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

21. 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.

22. Electroporation It is the most efficient method.
2.5 kV,200 ohms about 5 milli seconds required for bacterial cell.

24. Any More Questions?
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