We are currently trying to transform Bacillus licheniformis (environmental isolate) with plasmid pWH1520. Unfortunately, we barely get colonies growing on our plate regardless of the transformation protocol we used. We started with protoplast-based transformation before finally using electroporation but it didn't make any difference. Still no colonies growing and we have no clue why. We also read that some strains might be difficult to transform because of endogenous restriction system that rejects foreign DNA and that transforming B. licheniformis with unmethylated DNA can circumvent the problem. Do you think it might be the reason behind all those failed transformations or are there any critical steps we missed and therefore we might need to incorporate in our current protocol to make it work? Your feedbacks and suggestions are very much appreciated. Thank you.
Are you sure the promoters in your plasmid are compatible with this species?
I think so. pWH1520 is an E.coli-Bacillus shuttle vector with xylose promoter that should work in Bacillus (https://www.mobitec.com/products/vector-systems/bacillus-expression-systems/bmeg03/bacillus-megaterium-vector-pwh1520). Another feature like origin of replication should also be compatible with Bacillus in general, so the plasmid in theory can be maintained under selective environment.
If you think restriction enzymes are a problem you could try a crude protein extract of your bacteria and perform a restriction digest of your plasmid with exonuclease inhibitors before running a gel.
You could try expressing GFP or LacZ under the same promoter as the antibiotic selection cassette and then plating at a high density without antibiotic selection and screening if the other marker is detectable.
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