Material Required:
1. DH5α Host cells stock
2. Luria Bertani medium: The composition of the luria Bertani and other bacterial expression media is given in Table 37.2. For preparation of media dissolve the components in 1 liter of distilled water. Cover the top of the flask with cotton plug or aluminium foil and autoclave the solution at 121°C for 20 minutes. The various antibiotics or nutrient supplement should be added to the media when the temperature is less than 50°C (Figure 37.1 ).
3. 0.1 M Sterile MgCl2, 0.1M sterile CaCl2
Figure 37.1:Equipments and media required for sterilization and growth of bacterial expression system. (A)autoclave (B)autoclaved LB broth (C) E.coli grown in LB broth.
Methods :
1. Bacterial Culture- The growth stage of the bacteria has a significant impact for its ability to take up foreign DNA. The bacterium at log phase is more active and efficient to perform DNA damage and repair than stationery phase. As a result, it is preferred to use a bacteria of log phase for making competent cells for transformation.
2. Preparation of Competent Cell-Bacteria is incubated with divalent cation (Calcium chloride,Manganese chrloride or Rubidium chloride) for 30mins at 40C. During this process, cell wall of treated bacteria is swell and it gather factors required for intake of DNA docked on the plasma membrane. The whole process of E.coli competent cells preparation is as follows:
1. Inoculate single colony into the 100ml LB media and allow the cells to grow at 370C, 180rpm until
OD600 nm reaches to the 0.4-0.6.
2. Centrifuge the bacterial culture at 4000 rpm, at 4°C 10 min. Discard the supernatant.
3. Resuspend the cell pellet gently first in 1-2 ml and then in 10 ml of ice-cold 0.1 M MgCl2.
4. Centrifuge the bacterial suspension at 4000 rpm, at 4°C 10 min. Discard the supernatant.
5. Resuspend cells gently in 3.0 ml of ice-cold 0.1 M CaCl2.
6. Incubate the cell suspension on ice for additional 2hrs.
7. Centrifuge the bacterial suspension at 4000 rpm, at 4°C 10 min. Discard the supernatant.
8. Resuspend cells gently in 3.0 ml of ice-cold 0.1 M CaCl2 containing 10% glycerol and store in small aliquot (100µl) at -800C. The cells can be used for transformation (discussed in later lectures).