Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579.
An electro-transformation procedure was established for Bacillus cereus ATCC 14579. Using early growth-stage culture and high electric field, the ectroporation efficiency was up to 2 x 10(9) cfu microg(-1) ml(-1) with pC194 plasmid DNA. The procedure was tested with three other plasmids, of various sizes, replication mechanisms and selection markers, and the transformation efficiencies ranged between 2 x 10(6) and 1 x 10(8) cfu microg(-1) ml(-)(1). The effects of two wall-weakening agents on electroporation rates were also evaluated. The transformation rate that was reached with our procedure is 10(3) times higher than that previously obtained with members of the Bacillus genus with similar plasmids, and 10(6) times superior than that achieved with available protocols for B. cereus. The proposed method is quick, simple, efficient with small rolling circle plasmids and large theta replicating plasmids with low copy number per cell, and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols.
Turgeon
Nathalie
N
Institut universitaire de cardiologie et de pneumologie, Hôpital Laval, Université Laval, Quebec City, Québec, Canada.
Laflamme
Christian
C
Ho
Jim
J
Duchaine
Caroline
C
D001409:Bacillus cereus / Q000235:genetics*; D004269:DNA, Bacterial / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D010957:Plasmids; D014169:Transformation, Bacterial
D004269:DNA, Bacterial