Optimization of technical conditions for the transformation of Pediococcus acidilactici P60 by electroporation

• Published in: Plasmid Vol. 58; no. 1; pp. 44 - 50
• Main Authors: Rodríguez, M. Carmen, Alegre, M. Teresa, Mesas, Juan M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2007
• Subjects: DNA, Bacterial - genetics; Electroporation; Pediococci; Pediococcus - genetics; Pediococcus acidilactici; Plasmids; Transformation, Bacterial; Plasmids; Electroporation; Pediococci
• ISSN: 0147-619X; 1095-9890
• DOI: 10.1016/j.plasmid.2006.12.005

Previously reported techniques for the electrotransfer of foreign DNA into pediococci yield only a small number of transformants/μg 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) × 10 4 transformants/μg 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 20 kV/cm) and a reduction in lysozyme concentration during the preparation of electrocompetent cells (from 4000 to 2000 U/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 40 mM) also contribute to improved electrotransformation efficiency.