IGEM:Cambridge/2008/Turing Pattern Formation/BCSG Transformation Protocol

=B. subtilis Competent Cell Preparation and Transformation=

Competence development in B. subtilis is one of several stationary phase processes triggered by a nutritional downshift. Since the pioneering work of Anagnostopolous and Spizizen, a number of protocols for preparing competent B. subtilis cells have appeared. The following method, modified by Ron Yasbin from protocols developed in the Frank Young lab at Rochester, was used routinely in Stan Zahler's wonderful bacterial genetics course at Cornell. This protocol assumes that you use a spectrophotometer that accepts 16×125 mm test tubes. If your spectrophotometer, like mine, works only with cuvettes, simply increase the culture volume to 10 or 20 ml in a 250-ml Erlenmeyer flask.

1. Streak recipient strain on one-half of a Tryptose Blood Agar Base plate. Incubate overnight (18 hr) at 37°C.

2. Inoculate a few colonies into 4.5 ml of Medium A in a 16×125 mm test tube that lacks visible scratches. Mix the contents of the tube thoroughly. Read its optical density at 650 nm in the spectrophotometer. Adjust the OD650 to be 0.1-0.2, maintaining the volume at 4.5 ml.

3. Incubate at 37°C with vigorous aeration. Read the OD650 every 20 min, plotting OD650 against time on semi-log paper. After a brief lag, the OD should increase logarithmically—that is, they should fall on a straight line. Note the point at the culture leaves log growth—the graph points fall below the straight line. In B. subtilis genetics, this point is known as t0. It should take 60-90 minutes of incubation and occur at OD650=0.4-0.6.

4. Continue incubation for 90 minutes after the cessation of log growth (t90). Transfer 0.05 ml of this culture into 0.45 ml of pre-warmed Medium B in a 16×125 mm test tube. Set up one tube for each transformation you intend to perform, plus an extra for a DNA-less control.

5. Incubate the diluted cultures at 37°C with vigorous aeration for 90 min. At this point, the cultures should be highly competent.

6. Add 1 μg of DNA to the competent cells and incubate at 37°C with aeration for 30 minutes.

7. Plate aliquots of the transformed cells onto selective agar.

10× Medium A base: Yeast extract 10 g Casamino acids 2 g Distilled water to 900 ml Autoclave, then add: 50% glucose, filter-sterilized 100 ml

10× Bacillus salts (NH4)2SO4 20 g K2HPO4·3H2O 183 g KH2PO4 60 g Na+citrate 10 g MgSO4·7H2O 2 g Water to 1000 ml

Medium A Sterile water 81 ml 10× Medium A base 10 ml 10× Bacillus salts 9 ml Medium B Medium A 10 ml 50 mM CaCl2·2H2O 0.1 ml 250 nM MgCl2·6H2O 0.1 ml

References:
cells. J. Bacteriol. 121:296-304.
 * Yasbin, R. E., G. A. Wilson, and F. E. Young. 1975. Transformation and transfection in lysogenic strains of Bacillus subtilis: Evidence for selective induction of prophage in competent
 * Zahler, S. A. (personal communication)