Optimization of electrotransformation conditions for Propionibacterium acnes.
Propionibacterium acnes has been known to be involved in the pathology of acne. However, the definite mechanism in the development of acne and the inflammation are unknown. For P. acnes, a transformation method has not been established, although it is believed to be a basic tool for gene manipulation. This study attempted to develop a P. acnes transformation method by using electroporation. Various parameters were used to develop and optimize the transformation of P. acnes. Among them two factors were crucial in the transformation for P. acnes: one was the E. coli strain from which the plasmid DNA had been isolated and the other the growth temperature of P. acnes-competent cells. It was essential to prepare plasmid DNA from a dam(-) E. coli strain, ET12567. When plasmid DNAs isolated from the other E. coli strains such as JM109 and HB101 were tested, transformation efficiency was extremely low. When P. acnes cells were cultivated at 24 degrees C for competent cell preparation, transformation efficiency increased considerably. When plasmid DNA isolated from a dam(-) mutant strain of E. coli was used for transformation of P. acnes which had been grown at 24 degrees C, maximum transformation efficiency of 1.5 x 10(4) transformants per mug of plasmid DNA was obtained at a field strength of 15 kV/cm with a pulse time of 3.2 ms. This is believed to be the first report on the transformation of P. acnes which can be employed for gene manipulations including knock-out of specific genes.
Cheong
Dae-Eun
DE
Department of Biological Engineering, College of Engineering, Inha University, Incheon, South Korea.
Lee
Hae-In
HI
So
Jae-Seong
JS
D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D018274:Electroporation / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D010957:Plasmids; D011425:Propionibacterium acnes / Q000235:genetics* / Q000254:growth & development; D013696:Temperature; D014169:Transformation, Bacterial
D003470:Culture Media; D004269:DNA, Bacterial