CHAPTER 2 General materials and methods
2.18 Transformation of bacterial cells with DNA
Two different methods of transformation were used in the present study, depending on the type of plasmid DNA being transformed. For supercoiled plasmid DNA, the heat shock method (Section 2.18.2) of transformation was used. For ligation reactions, electroporation (Section 2.18.1) was used to transform bacteria. In order for the plasmid DNA to be taken up by the bacterial cells with each of these methods, the cells first have to be made competent.
For transformation by electroporation, cells were made competent using the glycerol method (Dower et al., 1988) (Section 2.18.1). Cells being transformed by the heat shock method were made competent using an adaptation of the CaCl2 method (Section 2.18.2) described by Cohen et al. (1972). Table 2.7 displays the E. coli cell strains used and the plasmids they were transformed with.
Table 2.7. Genotypes of E. coli cells transformed with recombinant plasmids
2.18.1 Transformation of competent E.coli (glycerol method) cells by electroporation Electroporation is a process allowing bacterial cells to take up free DNA. The mechanism involves exposure of the cells to a quick electric pulse, causing a type of structural rearrangement of the membrane, resulting in the dimpling of the cell membrane (Weaver, 1993). These dimples are thought to then form temporary hydrophobic pores ranging in diameter from 2 nm to 7 nm (Sambrook and Russell, 2001). Whilst the pores are open, ions and water-soluble molecules, including free DNA, are able to cross the membrane into the cytoplasm of the cell (Weaver, 1993).
Name Genotype Recombinant plasmid cells
were transformed with
E. coli JM103
endA1 glnV44 sbcBC rpsL thi-1 ∆(lac- proAB) F`[traD36 proAB+ lacIq
lacZ∆M15] pTS822; pMAL
E. coli JM109
endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ ∆(lac-proAB) glnV44 e14- [F`
traD36 pro AB+ lacIq lacZ∆M15]
hsdR17(rk-mk+)
pGEX4T1; pGEX4T1C18
E. coli BL21 F- ompT hsdSB (rB- mB-) gal dcm pGEX4T1; pGEX4T1C18
E. coli BL21(DE3)
F- ompT gal dcm lon hsdSB(rB-
mB-)λ(DE3
[lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) pGEX4T1; pGEX4T1C18 Turbo competent
E. coli (New England Biolabs)
F` proA+B+ lacIq ∆lacZM15/fhuA2 ∆(lac- proAB) glnV zgb-210::Tn10 (TetR) endA1 thi-1 ∆(hsdS-mcrB)5
pKK223-3-PfLDH;
pKK223-3-PvLDH
Preparation of competent cells for electroporation requires washing the cells with ice- cold water. Washing the cells at low temperature reduces the risk of arcing, which is the uneven transfer of charge across the cell-DNA suspension. Arcing occurs at high temperatures and at high ionic strength, and therefore washing with ice-cold water greatly reduces the number of ions present, in addition to keeping the cells at a reduced temperature. If arcing occurs, the efficiency of transformation is greatly reduced (Sambrook and Russell, 2001).
2.18.1.1 Reagents
2xYT media. Section 2.9.
Sterile ice-cold dH2O. dH2O was autoclaved and placed on ice until needed.
10% (v/v) glycerol. Glycerol (10 ml) was made up to 100 ml with dH2O and mixed thoroughly before autoclaving.
Super optimal broth (SOB) media. Section 2.9.
2.18.1.2 Procedure
An overnight culture (10 ml; 2xYT media) of appropriate E. coli cells was grown at 37˚C with shaking. Fresh 2xYT media (99 ml) was inoculated with 1 ml of the overnight culture and incubated at 37˚C with shaking until the culture reached an OD600 of 0.9. The culture was left on ice for 30 min before centrifuging (3 600 g, 10 min, 0˚C). The supernatant was discarded and the cell pellet was resuspended in ice-cold sterile dH2O (100 ml). The cells were centrifuged (3 600 g, 10 min, 0˚C) and resuspended in ice-cold sterile dH2O (50 ml). The cells were centrifuged (3 600 g, 10 min, 0˚C). The cell pellet was resuspended in ice cold sterile 10% (v/v) glycerol (2 ml) before centrifuging (3 600 g, 10 min, 0˚C). The cells were resuspended in ice cold sterile 10% (v/v) glycerol (1 ml). Aliquots (50 μl) were stored in sterile microfuge tubes on ice until required. DNA (1-2 µl) was gently mixed with freshly prepared competent E. coli cells (50 µl) and incubated on ice for 30 min. The DNA and cell mixture was pipetted into a pre-chilled, sterile electroporation cuvette (0.2 cm gap). A Bio- Rad Gene Pulser was used to apply one pulse of 25 microfarads, 2.5 kilovolts, and 200 ohms for approximately 4.8 milliseconds. Pre-warmed SOB media (300 μl) was gently mixed with the transformed cells. The cells and media were transferred to a sterile microfuge tube and
incubated at 37°C for 1 h. LB plates containing appropriate antibiotic were spread with the transformed cells (200µl) and incubated at 37°C overnight.
2.18.2 Transformation of competent E. coli cells (calcium chloride method) using heat shock
The mechanism behind the uptake of free DNA by cells with heat shock is not understood. The crucial step of the method involves briefly exposing competent cells to an elevated temperature. The temperature and length of exposure need to be accurate otherwise transformation efficiency is reduced (Sambrook and Russell, 2001). Once transformed, the cells are supplemented with a rich media to allow the cells to recover and begin expressing the antibiotic resistance marker, encoded by the plasmid. Competent E. coli cells were prepared using the modified method of Cohen et al., (1972). It is a highly effective method, typically yielding 5 X 106 – 2 X 107 transformed colonies per microgram of supercoiled plasmid (Sambrook and Russell, 2001).
2.18.2.1 Reagents
2xYT media. Section 2.9.
0.1 M CaCl2. CaCl2.2H2O (1.47 g) was dissolved in 100 ml dH2O and sterilised by autoclaving.
LB agar plates. Section 2.9.
Super optimal catabolizer (SOC) media. Section 2.9.
2.18.2.2 Procedure
Media (2xYT, 10 ml) was inoculated with one E. coli colony from an LB plate and left to grow at 37˚C with shaking overnight. The overnight culture was added to fresh 2xYT (90 ml) and left at 37˚C with shaking until an OD600 of 0.3-0.4. The culture was transferred to ice cold, sterile centrifuge tubes and left on ice for 10 min. The cells were pelleted (4500 g, 10 min, 4˚C). The supernatant was discarded and pellet was resuspended in ice-cold sterile 0.1 M CaCl2 (40 ml). The cells were pelleted as before, and resuspended in 0.1 M CaCl2 (2 ml). The cells were aliquoted (20 μl) into sterile 1.5 ml microcentrifuge tubes and stored on ice until use. The cells were used for transformation on the day of preparation.
Super coiled recombinant plasmid DNA (1 μl) was incubated with freshly prepared competent cells (20 μl) on ice for 30 min. Cells were heat-shocked at 42˚C for 90 s, and then immediately placed on ice for 2 minutes. Pre-warmed (37˚C) SOC media (80 μl) was added to the cells and gently mixed. The cells were then incubated for 1 h with shaking at 37˚C. LB agar plates containing appropriate antibiotics were then spread with the cells (50 μl), and then incubated overnight, inverted at 37˚C.