The process of making Escherichia coli cells competent for transformation. Essay
The process of making Escherichia coli cells competent for transformation., 494 words essay example
In the early stage of this practical, we had prepared Escherichia coli cells and we conducted protocols to make the cell be competent for transformation. E.coli was chose as many recombinant protein can be produced due to its simplicity to use, cheap, fit various expression vector, host strain and scientist already had the knowledge on its genetic characteristics. At the beginning, all bacteria cells we used were not competent cells and we need induced competency in it. Competent cells were those can take up DNA and competency can be induced artificially by chemical transformation or electro-transformation. In this practical, calcium chloride (CaCl2) is used to induce the E.coli cells to perform transformation. It disrupt the cell membrane and making it more permeable for uptake of DNA. Electroporation was not used because it needed skillful personnel and the cost was high. CaCl2 was used instead of rubidium chloride due to it less reactivity. Electroporation used electric pulse to pore on the cell envelope. Stanley Cohen, the scientist which developed the CaCl2 method in year 1972. He had successfully transformed R-factor and recombinant plasmids into E.coli using the calcium chloride method (Cohen et al, 1972). In this practical, few thing need to be concern to prevent failure in transformation. First, the time for centrifuge of cell suspension should be 5 minutes at 4 degreesC. Prolonged centrifuge will caused dead cell form sediment in the suspension cells and affect the transformation efficiency. Concentration of CaCl2 used was also important, 75mM of CaCl2 was used in this practical as this is the optimum concentration for competent cells to reach maximum transformation. The efficiency will decrease at 100mM of CaCl2 (Li et al, 2010). The cells also grew until OD550 was between 0.3-0.5. This is because at that level, the cells had reached maximum log phase and the chances of transformation will be increased as the cells divided most rapidly (Li et al, 2013). At OD550 exceeds 0.8, the cell reached stationary phase and stop dividing. Cell started to death and not suitable for transformation.
During the chemical transformation process, after centrifuged, the supernatant was discarded and pellet resuspended in CaCl2 with glycerol. The addition of glycerol is to reduce contamination (Li et al, 2010). Glycerol also acted as cryopreservative. During freezing process, water turned to ice and it might damage the cell. The outer part of the cell freeze before the intracellular ice begun to form (Wood, 1980). When ice formed, water will transferred out from the cell to the solution. Excess water which remained in the cells will caused damage to the cells' contents due to ice crystal formation and recrystallization during thawing (Mazar et al, 1972). Glycerol can minimize the detrimental effects of ice crystal formation. The competent cells were aliquoted into Eppendorf tubes and quick-freezed them in liquid nitrogen which stored at -70degreesC before they were used for transformation. The competent cell can be stored at -70degreesC for 15 days and it would not affected the transformation efficiency (Tu et al, 2004).