Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation.
OBJECTIVE
The ability to transform Vibrio spp. is limited by the extracellular nuclease that their cells secrete. The reported transformation efficiency of this organism is 10(2)-10(5) transformants per microgram DNA. We tried different buffers and conditions, aiming to elevate its transformation efficiency.


RESULTS
MgCl(2) and sucrose are often included in the washing and/or electroporation buffers to stabilize the cell membrane. However, Mg(2+) is required for production and activity of the extracellular nuclease. A simple electroporation buffer lacking Mg(2+) was found to increase transformation efficiency dramatically, to levels 50-fold more than the buffers containing Mg(2+). To maintain the stability of the cell membranes, Mg(2+) was replaced with high concentrations of sucrose, from 272 to 408 mmol l(-1). With the new buffers, the transformation efficiency of Vibrio parahaemolyticus was increased to 2.2 x 10(6) transformants per microgram DNA.


CONCLUSIONS
Mg(2+) in the buffer adversely affected transformation of V. parahaemolyticus by electroporation. The cell membranes of vibrio can be stabilized by high concentration of sucrose when Mg(2+) is absent.


CONCLUSIONS
A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg(2+) can be used for other nuclease-producing organisms.
Wang
H
H
Department of Food Science, Canadian Research Institute for Food Safety, University of Guelph, Guelph, ON, Canada.
Griffiths
M W
MW
D000818:Animals; D049872:Bivalvia / Q000382:microbiology; D002021:Buffers; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D003851:Deoxyribonucleases / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000032:analysis; D013395:Sucrose / Q000032:analysis; D014170:Transformation, Genetic; D014736:Vibrio parahaemolyticus / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification
D002021:Buffers; D003470:Culture Media; D004269:DNA, Bacterial; D013395:Sucrose; D003851:Deoxyribonucleases; D008274:Magnesium