Competent Listeria Cells - (Jan/11/2008 )
I am a technician, currently working on a project involving Listeria monocytogenes. At the moment my post-doc and I are spending a lot of time preparing competent Listeria cells, following a published protocol (I'm at home right now so I don't have the reference unfortunately).
Essentially, the cells are grown in Tryptone Soya Broth containing 20% sucrose overnight, subcultured, then washed in a HEPES/sucrose solution. However, neither of us are sure why such a high concentration of sucrose is needed. The paper we are following specifically states that the sucrose is not needed for osmotic stress, so why is it there?
Does anybody have any suggestions? It's probably something really obvious which we have both missed due to our brains being addled from washing and spinning the cells all day!
One is tempted to say "because the first person to do it did it that way." People dislike change, especially change to things that are working. If you figure the answer to this out, I'd like to know as well.
That is very true! If I find an answer, I will definitely post it here.
I looked into this a bit. The sucrose apparently comes from previous transformation protocols which used spheroplasts of gram positive bacteria and plants, and was an osmoprotectant. I still haven't found the place where 272 mM sucrose was first used, but it seems to have been common in 1988 and subsequently for work with bacteria, except for E. coli, for which water washing was, and still is, used. I did find some places where 500 mM sucrose solutions were diluted 2:1 with culture medium to give a 250 mM sucrose final concentration. Experimentation may improve your results. FYI I have found that with my favorite organism, trypsin treatment of the cells prior to electroporation dramatically improves transformation efficiency, presumably removing membrane nucleases.