Last week I did an experiment in parallel with one of the post-docs. Her preparations of competent H. influenzae had not been very competent at all, and we wanted to check if there was a problem with her procedure. We both produced cells that were reasonably (not excellently) competent, so we concluded that her past problems were not due to faults in her procedure.
In recent posts I've been describing my preliminary steps to finding a way to induce E. coli's 'cryptic' competence genes, and this and the above have started me thinking about the procedure we use for H. influenzae. It's a simple sprocedure: cells growing in a rich medium called sBHI are abruptly transferred to a starvation medium called M-IV. After about 90-100 minutes in this medium the cells are as competent as they are going to get. I deally this means that all ofthe cells are competent, each ready to take up a few hundred kb of chromosomal DNA.
This procedure was developed about 45 years ago, by more-or-less systematic fiddling with growth conditions. It hasn't been altered, simply because nobody has had a strong reason to bother trying. But now we know much more about what is going on when competence genes are induced, and we should be able to improve on this procedure. Improvements would certainly be useful, especially for the post-doc's ongoing survey of competence in 'wild' strains of H. influenzae. Being able to improve the induction procedure is also a good test of whether we really do understand how the genes are regulated.
What should we try? Adding cAMP should take the place of carbohydrate starvation, activating CRP to make its contribution to expression of CRP-S promoters. If we're right that nucleotide starvation is needed to induce sxy translation, we would like to try simpler ways to mimic this; I wonder if there's a specific inhibitor pf purine (or pyrimidine) synthesis? But this won't be much use if the cells are still in rich medium, as they are getting their nucleotide precursors from the medium not from de novo synthesis. Maybe we could simulate nucleotide starvation another way (slowing polymerase with an antibiotic resistance mutation? as we suggested in one of our grant proposals???).
If at first you don't succeed, put it all back they way it was and keep lumbering along
11 hours ago in The Phytophactor