This question may be a little naive, but I've never seen anyone use blue white screening in a gram-positive bacteria before and wanted to know why! I know you need lacZM15 mutants in E. coli, but I was thinking about using lacZ as a reporter to look at expression driven by an enterococcus promoter in the context of different mutant backgrounds. I thought it would be good to be able to initially screen a lot of them qualitatively on a plate with X-gal.
The main reason is: it doesn't work very well. Assuming you're using an Enterococcus species, their cell wall doesn't let much across passively. For E. coli you can get obvious blue colonies at 20 µg/mL X-gal. At 200 µg/mL in Enterococcus faecalis you will begin to see a faint blue-green tint to the colonies if lacZ is being expressed from a strong promoter, you let the colonies sit at 4° for a while and you squint a little with the plate against a white background.
Some other reasons:
1. E. coli lacZ has poor codon usage for firmicutes.
2. The full length lacZ gene is pretty big (~3 kb) so you bump up the plasmid size quite a bit just by including it.
3. Gram positives have much worse electroporation efficiency compared to E. coli so it's difficult to build large promoter clone libraries for screening. (It's been done but it doesn't sound fun.)
Thanks for the perfect answer Alexandra Johnson ! I'll look for a different reporter gene :)
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