Optimization of electroporation procedure to transform B. polymyxa SCE2 and other nitrogen-fixing Bacillus

Alexandre Rosado, Gabriela F. Duarte, Lucy Seldin

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16 Scopus citations

Abstract

An efficient method for genetic transformation of different nitrogen-fixing Bacillus polymyxa strains by electroporation is presented. Various parameters on plasmid transformation of b. polymyxa SCE2 with plasmid pC194 were investigated to enhance transformation efficiency: voltage, buffer strength and type of electroporation buffer, plasmid DNA concentration, purity of plasmid preparation and plasmid source. Electroporation of B. polymyxa SCE2 resulted in a transformation efficiency as high as 3.2 × 105 transformants/μg of pC194 when the optimized procedure was used. Stability of different plasmids (pE194, pBD64, pBC16 and pFT30) transformed to B. polymyxa SCE2-43 and/or SCE2-43 was analysed. pE194 showed high frequency of curing while pFT30 and pBC16 were stable after 10 transfers in non-selective medium. Plasmid DNA isolated from transformants had not undergone detectable deletions. Although the electroporation protocol presented here can be used to transform B. azotofixans, lower efficiencies of transformations were achieved. No transformants were detected in other nitrogen-fixing strains belonging to B. macerans. © 1994.
Original languageEnglish (US)
Pages (from-to)1-11
Number of pages11
JournalJournal of Microbiological Methods
Volume19
Issue number1
DOIs
StatePublished - Jan 1 1994
Externally publishedYes

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