A22 Transformation of E. coli with Plasmid

Purpose

To demonstrate the techniques for transforming E. coli with a plasmid.

Prior Work

A ligated vector and a source of cells to transform (such as bacteria) must be available.

Materials

Background

Transformation is a permanent genetic change induced in a cell following the incorporation of foreign DNA. Transformation is a pivotal step in biotechnology. Only a small fraction of the cells treated to undergo transformation is able to be transformed. Only a tiny fraction of the passenger DNA molecules is successful at transformation. Several transformation procedures are available; the one described here uses calcium chloride.

Selection of the bacteria depends upon the experiment, but strains of E. coli are often used. There is a biohazard potential while working with bacteria.

Procedure

  1. Prepare an "overnight" culture of the bacteria to be transformed. See lesson A-09 for suitable procedures. All of the equipment that comes into contact with bacteria is autoclaved routinely. All bacteria are killed before disposal. Sterile transfer technique will be used throughout. All of the vessels shown have been sterilized. Wear gloves at all times. Treat the bacteria as if they are hazardous. Work in a laminar flow hood.
  2. Obtain a sterile 1-mL aliquot of 20% glucose. Flame the opening of the glucose bottle and cover immediately. Prepare a sterile 250-mL Erlenmeyer flask closed with a sterile cotton plug. Remove the plug. Flame the opening of the flask. Add the glucose to the flask, flame the flask, and replace the sterile plug. Add 1 mL of inoculum to the L broth.
  3. Remove 1-mL aliquots periodically and transfer to a disposable spectrophotometer cell.
  4. (Place the pipet tip with trash that will be autoclaved prior to disposal.) Adjust the wavelength to 550 nm and set the absorbance to zero (100% transmittance). Place the cell into the spectrophotometer, cover and read the absorbance. Dispose of the aliquot and the spectrophotometer cell with material that will be autoclaved.
  5. (Some workers prefer to use a special flask with a sidearm tube that may be inserted directly into a spectrophotometer. These costly flasks are somewhat fragile, but allow frequent checking of the optical density without loss of material or risk of contamination. Tilt the flask so that the side arm fills with culture. Insert the sidearm in the spectrophotometer. Cover the flask with opaque material such as aluminum foil to eliminate stray light. Measure the optical density.)
  6. Once the desired extent of growth is reached, chill the culture on ice for at least 10 minutes.
  7. The desired number of bacteria is about 5 x 107 cells/mL. A curve relating optical density at 550 nm and number of cells is used and must be obtained for each strain of bacteria. The best transformation efficiencies occur when the bacterial culture is in the logarithmic growth phase when heated with calcium chloride, and the cells maintained at 4oC for 0.5-1.0 day after this treatment. Transfer the culture to suitable centrifuge tubes (e.g., Oak Ridge tubes). Cover. Centrifuge at 4,500 g for 5 minutes at 4oC.
  8. Note the volume of supernatant fluid. Return the supernatant fluid to the culture flask for autoclaving. Add a volume of sterile, ice cold 50 mM CaCl2 and 10 mM Tris (pH = 8.0) equal to half of the volume of supernatant fluid just discarded. Tighten the tube caps. Resuspend the cells using gentle agitation. Set the mixture on ice for 15 minutes. Centrifuge at 4,500 g for 5 minutes at 4oC. Note the volume of supernatant fluid. Pour the supernatant fluid into the original culture flask for autoclaving.
  9. Add 50 mM calcium chloride/10 mM Tris (pH = 8.0, 1/15th the volume of supernatant just discarded) to each tube. Tighten the caps. Resuspend using gentle agitation. Store for between 12 and 24 hours (overnight) at 4oC. A loose pellet forms from overnight storage. Resuspend this pellet by gentle agitation. Remove 200-µL aliquots of resuspended bacteria. Add these aliquots to chilled, labelled 1.5-mL microcentrifuge tubes. The cells are now competent to accept plasmid DNA. Add an aliquot of 40 ng plasmid DNA dissolved in up to 100 mL of 1x TE buffer or water. Store on ice for 30 minutes.
  10. Add the mixture containing the plasmid to one tube of competent cells. The basis of this procedure is to have one transforming DNA molecule per cell. Wait a full 30 minutes. Heat shock the cells at 42oC for 2 minutes. Add 1 mL of L broth. Transfer to a bath at 37oC. Incubate for 30-60 minutes. The transforming DNA -- nearly always as a part of the vector DNA component -- will include genetic information that imparts specific antibiotic resistance. After transformation, the bacteria will be grown in a broth containing antibiotic. Only those bacteria that have been transformed successfully should survive in the antibiotic broth. The duration of the 37oC incubation depends upon which antibiotic resistance gene the plasmid vector expresses.
  11. Remove aliquots and place on plates. Dip a glass streaking rod in 70% aqueous ethanol. Keep ethanol and flame a safe distance apart. Flame the rod, then use it to spread the moisture evenly on the surface of the agar. Leave the dishes slightly ajar to dry. Seal the plates with a strip of Parafilm2. Place the plates in an incubator at 37oC. Invert the plates so that condensing moisture will not drip onto the colonies on the surface of the agar. Leave the plates overnight.
  12. Medium selection depends upon the plasmid used. The medium contains an antibiotic and selects for the transformed bacteria. Selection of the antibiotic will depend upon the nature of the antibiotic resistance imparted by the vector. Tetracycline and ampicillin resistances are often employed.
  13. Several controls are needed. The transformed bacteria should be grown on plates with antibiotic. The plasmid vector should be cultured on its own. To do this, process the plasmid through the ligation procedure with all additions except for passenger DNA. A transformation control consists of a plasmid (without insert, that is, without ligation) and competent cells.
  14. If too many bacteria are plated, they will overwhelm the antibiotic and grow in spite of its presence. For this reason, control plates of non-transformed bacteria should be prepared to check that only transformed bacteria are able to survive under the experimental conditions.

Going Further

The appropriate treatment of the plates on which the colonies form depends upon the antibiotic sensitivity being expressed. The transformation experiment is complete. The individual colonies -- mostly clones -- can be studied by miniprep (A-27) and restriction analysis (A-28) or by colony lifts (A-24) and colony hybridization (A-26).

References