Optimization of technical conditions for the transformation of Pediococcus acidilactici P60 by electroporation.
Previously reported techniques for the electrotransfer of foreign DNA into pediococci yield only a small number of transformants/mug DNA, especially when using undomesticated strains. This study reports an improved protocol for the electrotransformation of pediococci, based on trials using Pediococcus acidilactici P60 and the plasmid pRS4C1. The improved protocol yields from 2 to 3 log units more transformants than the previously reported methods, with up to (9.1+/-1.3)x10(4) transformants/mug of foreign DNA under the best conditions identified. The most important modifications proposed are an increase in electric field strength during electroporation (from 12.5 to 20kV/cm) and a reduction in lysozyme concentration during the preparation of electrocompetent cells (from 4000 to 2000U/ml): together, these two modifications greatly improve transformant yield. In addition, increasing cell culture time (from OD(600nm)=0.6 to OD(600nm)=1.0-1.2) and increasing dl-threonine concentration in the growth medium (from 20 to 40mM) also contribute to improved electrotransformation efficiency.
Rodríguez
M Carmen
MC
Departamento de Fisiología Vegetal, Escuela Politécnica Superior, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
Alegre
M Teresa
MT
Mesas
Juan M
JM
D004269:DNA, Bacterial / Q000235:genetics; D018274:Electroporation; D010376:Pediococcus / Q000235:genetics*; D014169:Transformation, Bacterial
D004269:DNA, Bacterial