title: Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation.
abstract: 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.
authors.affiliation: Department of Food Science, Canadian Research Institute for Food Safety, University of Guelph, Guelph, ON, Canada.
authors.forename: H
authors.initials: H
authors.lastname: Wang
authors.forename: M W
authors.initials: MW
authors.lastname: Griffiths
mesh_terms: 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
chemical_list: D002021:Buffers; D003470:Culture Media; D004269:DNA, Bacterial; D013395:Sucrose; D003851:Deoxyribonucleases; D008274:Magnesium