Electroporation Lactic acid Bacteria - Problems establishment/reproducibility (Jan/31/2008 )

Posted: Jan 31 2008 10:39 am Post subject: Electroporation Lactic Acid Bacteria

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Hi!

I have some troubles with the transformation of Lactic Acid Bacteria.

I want to transform a vector based on the construct pNW33N (It should be applicable for a wide range of gram positive) into Lactobacillus reuteri and Pediococcus acidilactici.

So far I tried several protocols (Caldwell et al. 1996 - washing with sorbitol/glycerol; Aukrust et al. 1995 - washing with Polyethylenglycol or Sucrose; Rodriguez et al. 2007 - washing with Sucrose and Lysozme treatment) to make electrocompetent cells.
However, neither of them works for L. reuteri. Using the protocols from Aukrust and Rodriguez with P. acidilactici i sometimes get clones and sometimes it doesn't work.

So maybe somebody of you has any ideas, hints, protocols to establish a good tranformation protocol for both strains or at least to get reproducible results for Pediococcus ...?

I try and i try and ... i'm getting more and more despaired of it ....

Thx waikki

Your electroporation may be working, but the DNA may not be. Think about restriction systems present in these bacteria. If your DNA is cut up, it won't transform. You may need to engineer out cut sites after you find out what restriction systems are present in these organisms. If you are lucky, there is a commercial methylase which can be used to treat the DNA prior to transformation.