Preparation of competent cells

introduction and protocol for preparation of competent cells

1. PREPARATION OF COMPETENT CELLS FOR TRANSFORMATION BY KANCHAN YADAV MSC AGRIL. BIOTECHNOLOGY ,1 st YEAR DR. RAJENDRA PRASAD CENTRAL AGRICULTURE UNIVERSITY

2. Introduction of dna into a host cell – two key problems Must be able to physically cross the cell membrane Once inside the new host cell , our DNA must be able to replicate

3. TRANSFORMATION Transformation  is the genetic alteration of a cell resulting from the direct uptake and incorporation of  exogenous genetic material  from its surroundings through the  cell membrane (s). For transformation to take place, the recipient bacteria must be in a state of  competence , which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

4. COMPETENT CELLS Bacterial cells that are able to take up DNA from the environment are called competent cells. In the laboratory, bacterial cells can be made competent and DNA subsequently introduced by a procedures.

5. METHODS OF TRANSFORMATION USE OF CELLS WHICH ARE NATURALLY COMPETENT EG . BACTERIA Bacillus subtilis CALCIUM TREATMENT OF CELLS TRANSFORMATION OF PROTOPLASTS ELECTROPORATION OF CELLS/ PROTOPLASTS Using high voltage shocks for a fraction of a second to make membrane more permeable to DNA

6. METHODS OF TRANSFORMATION MICROPROJECTILE BOMBARDMENT Bombardment of intact cells with metal particles coated with DNA INFECTION OF CELLS WITH VIRUSES MICROINJECTION of DNA directly into a cell nucleus

7. HEAT SHOCK TRANSFORMATION Heat shock transformation uses a calcium rich environment provided by calcium chloride to counteract the electrostatic repulsion between the plasmid DNA and bacterial cellular membrane. A sudden increase in temperature creates pores in the plasma membrane of the bacteria and allows for plasmid DNA to enter the bacterial cell.

8. MATERIAL REQUIRED Buffers and solutions 1) CaCl2.2H2O ( 1M) 2) Standard transformation buffer(TFB) 3) MgCl2 – CaCl2 solution 4) Ice cold Media 1) LS or SOB medium for initial growth of culture. 2) SOB agar plates containing 20 mM MgSO4 and the appropriate antibiotic 3) SOC medium

9. MATERIAL REQUIRED contd. Nucleic Acids and Oligonucleotides Plasmid DNA Centrifuges and Rotors Sorvall GSA rotor or equivalent Equipment Polypropylene tube (50mL), chilled in ice Polypropylene tubes Water bath preset to 42 C

10. PROCEDURE 1. Inoculate the E. coli culture into the LB medium and incubate at 37 °C for 24 h with vigorous shaking at 180 rpm. 2. Aliquot 0.5 ml of the grown culture into 50 ml of LB in a 200-ml conical flask. Prewarm the broth to 37 °C. 3. Incubate at 37 °C with shaking at 180 rpm. 4. Monitor the growth regularly till the OD600 reaches to 0.35–0.4. 5. When suitable growth has been reached, chill the culture on ice.

11. PROCEDURE CONTD. 6. Transfer the culture to an autoclaved centrifuge tube and collect the cell pellets by centrifugation at 6000 rpm for 5 min at 4 °C. Discard the supernatant. 7. Resuspend the cell pellets in 20 ml of an ice-cold 50-mM CaCl2 solution. Incubate the resuspended cells on ice for 20 min. 8. Collect the cell pellets by centrifugation at 6000 rpm for 5 min at 4 °C. 9. Resuspend the cells with 2.5 ml of ice-cold 50-mM CaCl2. Optionally, if required to store the competent cells for a longer period, resuspend the cells with 2.5 ml ice-cold 50-mM CaCl2 containing 10 % glycerol. 10. Use 100 μl of the prepared competent cells for transformation. 11. Dispense the competent cells into aliquots of 100 μl and store them at − 80 °C for further use.

12. CONCLUSION Non-commercial preparations should normally give 10 6  to 10 7 transformants per microgram of plasmid; a poor preparation will be about 10 4 / μg or less , but a good preparation of competent cells can give up to ~10 8   colonies per microgram of plasmid . Protocols, however, exist for making supercompetent cells that may yield a transformation efficiency of over 10 9 .

13. CONCLUSION CONTD. The chemical method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native  exonuclease  enzymes rapidly degrade linear DNA. In contrast, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA. The transformation efficiency using the CaCl2 method decreases with plasmid size

14. Precautions 1. CaCl2 is a hazardous material for skin, eyes and the respiratory system and may cause burns. Hence, use gloves while using the same. 2. Avoid thawing of cells before use. 3. Keep cells on ice not longer than 3 h; do not use cells again that have been on ice. 4. You may stock-freeze the competent cells in liquid nitrogen. The stock-freezing might 5. Keep cells viable for a longer period, but it decreases the transformation efficiency by at least a factor of ten.

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