Microbiologic techniques

E. coli was grown in liquid LB medium or on LB agar (1.5% agar) plates at 37°C. Antibiotic concentrations for E. coli were as follows: ampicillin sodium salt 100 µg/ml, kanamycin suphate 50 µg/ml, tetracycline hydrochloride 10 µg/ml, and chloramphenicol 25 µg/ml.

4.2.1.2. Growth of S. aurantiaca

S. aurantiaca was grown in liquid Tryptone medium or on Tryptone agar (1.5% agar) plates at 32°C.

Antibiotic concentrations for S. aurantiaca were as follows: streptomycin sulphate 125 µg/ml, kanamycin sulphate 50 µg/ml.

4.2.1.3. Heat shock induction of S. aurantiaca

S. aurantiaca was grown in liquid Tryptone medium at 28°C until a log phase was reached (1.6 × 10⁸ cells/ml). Then the culture was shifted to a 38°C water bath with vigorous shaking.

4.2.1.4. Spore induction in S. aurantiaca by indole

S. aurantiaca was grown in liquid Tryptone medium at 28°C until a late log phase was reached (2.2 × 10⁸ cells/ml). Then indole (1 M in 100% ethanol) was added to the culture to a final concentration of 0.5 mM.

4.2.1.5. Fruiting body formation of S. aurantiaca

S. aurantiaca was grown in liquid Tryptone medium at 32°C until a log phase culture was reached (2

× 10⁸cells/ml). Cells were harvested by centrifugation at 4,000 × g for 15 min at 4°C. The cell pellet was washed twice with 10 mM HEPES, 0.5 mM CaCl2 (pH 7.2) buffer and then resuspended in the same buffer to a final concentration of 4 × 10¹⁰ cells/ml. 5-10 µl of the cell suspension was spotted on water agar plates. After drying, the plate was incubated at 32°C for 24 hours or longer. To analyse the temperature dependency of fruiting body formation, the assay was performed at different temperatures (30°C, 32°C, 35°C, 37°C, and 39°C) synchronously and the fruiting body formation was controlled after different time periods.

4.2.1.6. Preservation of E. coli and S. aurantiaca cultures

E. coli was grown to a log phase and 0.8 ml of the culture was transferred to a sterile tube and mixed with 0.2 ml of sterile glycerol. The culture was preserved at -80°C.

S. aurantiaca was grown to a log phase and 1 ml of the culture was transferred into a sterile tube and frozen with liquid nitrogen; or 1.5 ml of the culture was transferred to a sterile tube and spun down at 4,000 × g for 5 minutes. The cell pellet was resuspended in 0.4 ml liquid Tryptone medium containing 25% glycerol. The culture was preserved at -80°C.

4.2.1.7. Germination of S. aurantiaca spores

S. aurantiaca fruiting body formation was performed on water agar plates with filter papers. After the fruiting bodies matured on the filter papers, the fruiting-body containing filter papers were dried by incubating

with Silicon gel for one month at room temperature in a closed desiccator. The filter papers were then transferred on Tryptone agar plates (upside down) and incubated at 32°C for two days.

4.2.1.8. Electroporation of E. coli

Electrocompetent cells were prepared with 1 litre of E. coli culture in LB medium, which was inoculated with 1/100 volume of fresh overnight culture and incubated at 37°C with vigorous shaking until the OD600 value reached 0.5 to 1.0. After chilling down on ice for 30 min, cells were harvested by centrifugation at 4,000 × g for 15 min at 4°C. The cell pellet was washed once with 1 litre of cold H2O, once with 0.5 litre of cold H₂O and once with 20 ml of cold 10% glycerol. After resuspending the cells in cold 10% glycerola to a final volume of 2 to 3 ml, they were dispensed into 40 µl portions, frozen in liquid nitrogen and stored at -80°C.

For electroporation, 10 to 50 ng DNA dissolved in 1 to 5 µl of a low ionic strength buffer was mixed with the thawed electrocompetent cells in an eppendorf tube and the tube was kept on ice for about 1 min. The cell-DNA mixture was then transferred to a cold electroporation cuvette (Gap distance is 1 mm). Electroporation was performed with the BioRad GenePulser with a field strength of 12.5 kV/cm, an electric capacity of 25 µF and a resistance of 200 Ω. After electroporation, 1 ml of LB or SOC medium was added into the electroporation cuvette immediately. The cell suspension was transferred to a test tube and incubated at 37°C with vigorous shaking for one hour. Then the cells were spreaded onto LB agar plates containing appropriate antibiotics.

SOC medium:

Tryptone 2%

Yeast extract 0.5%

NaCl 10 mM

KCl 2.5 mM

MgCl2 10 mM

MgSO₄ 10 mM

Glucose 20 mM

4.2.1.9. Electroporation of S. aurantiaca (Stamm et al., 1999)

The electrocompetent cells were prepared freshly with a log phase culture of S. aurantiaca. The cells were harvested by centrifugation at 4,000 × g for 15 min at 20°C. The cell pellet was washed once with an equal volume of 5 mM HEPES, 0.5 mM CaCl₂ buffer, pH 7.2, at room temperature, following with 1/2 volume of the same buffer. The cells were then resuspended in the same buffer to a final concentration of about 4 × 10¹⁰ cells/ml. About 0.1 to 0.5 µg DNA was mixed with 40 µl competent cells. Electroporation was performed with the BioRad GenePulser with a field strength of 8.5 kV/cm, an electric capacity of 25 µF and a resistance of 200 Ω. Thereafter, the S. aurantiaca cells were grown in 50 ml of liquid Tryptone medium without selective antibiotic at 32°C for 20 hrs. The cells were harvested by centrifugation at 4,000 × g for 15 min at 4°C and resuspended in 2 ml of 0.1 M HEPES, pH 7.2, 10 mM CaCl2 buffer. 0.1-0.5 ml of the cell solution was mixed with 3 ml of pre–warmed (42°C) soft agar Tryptone medium (0.75% agar, containing appropriate antibiotics) and then placed on a Tryptone agar plate containing appropriate antibiotics. The plates were incubated at 32°C for 5 to 7 days.

4.2.1.10. White-blue colony selection of E. coli

Some cloning vectors (e.g. pBC SK+ and pBluescript SK-) carry the regulatory sequences and the first 146 amino acids of the coding region of the ß-galactosidase gene. A polycloning site is embedded in the coding region. After transferring into E. coli host cells that code for the carboxy-terminal portion of ß-galactosidase, the

active ß-galactosidase will be obtained due to the complementation of the N- and C- portions of ß-galactosidase.

Blue colonies will appear in the presence of chromogenic substrate X-gal. When foreign DNA is inserted into the multiple cloning site of the vector, the N–terminal portion will be disrupted and white colonies appear.

To select such recombinant clones, bacteria cells were spreaded on LB agar plates that were placed by 40 µl of X-gal stock buffer and 40 µl of IPTG stock buffer.

4.2.1.11. Expression of fusion protein in E. coli M15 and GI698

E. coli M15 cells carrying pQE-expression plasmid were grown in LB medium at 37°C overnight. A fresh culture was inoculated with the overnight culture at a ratio of 1:50 and then incubated at 37°C with vigorous shaking until a log phase (OD₆₀₀ = 0.7-0.9) was reached. IPTG was added to the culture to a final concentration of 1-2 mM. The maximum expression of fusion protein was obtained after about 3-5 hrs.

When using E. coli GI698 to express the fusion protein, the cells carrying pQE-expression plasmid were grown in 10 ml of RM medium at room temperature overnight. After inoculation with the overnight culture at a ratio of 1:20, the fresh culture in Induction medium was grown at room temperature with vigorous shaking until a log phase (OD550 = ca. 0.5) was reached. IPTG and tryptophan were added to the culture to a final concentration of 2 mM and 100 µg/ml, respectively. The maximum expression of fusion protein was obtained after about 5 hrs.

RM medium (Invitrogen):

Na₂HPO₄ 0.6%

KH2PO4 0.3%

NaCl 0.05%

NH₄Cl 0.1%

Casamino acids 2%

MgCl₂ 0.0095%

PH 7.0

Induction medium (Invitrogen):

Na₂HPO₄ 0.6%

KH₂PO₄ 0.3%

NaCl 0.05%

NH4Cl 0.1%

Casamino acids 0.2%

MgCl₂ 0.0095%

PH 7.0

Tryptophan solution:

Tryptophan 10 mg/ml in H₂O