An improved protocol for electroporation of Oenococcus oeni ATCC BAA-1163 using ethanol as immediate membrane fluidizing agent.
OBJECTIVE
To finalize an effective and reproducible electroporation procedure to transform Oenococcus oeni ATCC BAA-1163 strain.


RESULTS
The vector pGID052 was selected to optimize the electroporation procedure. Transformation efficiency was 5.8 x 10(3) per microg of DNA. Transformation was improved when competent cells were prepared with exponential phase cultures; optimum electroporation parameters were an electric pulse of 125 kV cm(-1), under a resistance of 200 omega and the presence of 10% (v/v) ethanol in the electroporation buffer (EPB).


CONCLUSIONS
An effective protocol to transform O. oeni ATCC BAA-1163 strain by electroporation has been obtained by addition of ethanol to the EPB. A heterologous expression was obtained in O. oeni ATCC BAA-1163 by introducing a recombinant vector encoding a truncated form of ClpL2 protein.


CONCLUSIONS
This is the first report of a successful electroporation of O. oeni ATCC BAA-1163. The major improvement was the addition of ethanol to the EPB, which has never been reported before as means of enhancing the incorporation of foreign DNA molecules into prokaryote cells by electroporation. This method constitutes a useful tool for the genetic study of this lactic bacterium.
Assad-García
J S
JS
Université de Bourgogne - IUVV Jules Guyot, Laboratoire de Recherche en Vigne et Vin, Dijon, France.
Bonnin-Jusserand
M
M
Garmyn
D
D
Guzzo
J
J
Alexandre
H
H
Grandvalet
C
C
D001426:Bacterial Proteins / Q000235:genetics; D002462:Cell Membrane / Q000187:drug effects*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D000431:Ethanol / Q000494:pharmacology*; D006093:Gram-Positive Asporogenous Rods / Q000187:drug effects* / Q000235:genetics*; D010957:Plasmids / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000431:Ethanol