Methods of Transformation/Transfection

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 cellsare 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.

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