Transformation of Bacillus stearothermophilus No. 236 by Changing incubation temperature after electroporation

Bacillus stearothermophilus No. 236 isolated from the soil is a strong xylan degrader producing all the xylanolytic enzymes. However, the strain was discovered to be highly intractable to its transformation. In the present study, we have developed a reliable method for transformation of B. stearothermophilus No. 236 by a systematic examination of several factors which might have an influence on the efficiency of electrotransformation. Notably, we found that the most critical factor influencing the transformation efficiency (TE) was the incubation temperature after pulsing, with its optimum incubation of 37°C. At 50°C, the optimum growth temperature of the B. stearothermophiIus strain, the transformants could not be obtained at a recognizable level. The combination of field strength of 7.5 kV/cm along with pulse duration of 10 msec (resistance of 400 Ω and capacitance of 25 μF) was shown to be the best electrical parameters at the incubation temperature of 37°C. A higher TE was obtained when the cells were harvested at an early-exponential phase. Twenty percent of PEG-8000 in a suspension buffer and an addition of 0.1% glycine in the growth medium resulted in about 4-fold and 3-fold increases in TE, respectively. We also found that the plasmid DNA which had been cycled through the host B. stearothermophilus cells enhanced TE by one order of magnitude higher. Under the presently described conditions, 2.5x 10⁵ transformants per μg DNA was attained.