The word around the lab is that the standard technique for transforming H. influenzae cells with plasmid DNA hasn't worked for anyone in years, so I'm testing it myself. It always worked OK for me, but I haven't done it in a long time. The technique is simple: cells are made naturally competent in the usual way (starvation in MIV medium) and incubated with plasmid DNA. The plasmid DNA is efficiently taken up into the periplasm, but it can't get across the inner membrane because that translocation needs a DNA end and the plasmid is circular, The cells are then given an osmotic shock (30% glycerol for 10 minutes), which somehow drives the plasmid into the cytoplasm.
Why do this now? Lord knows I have lots of other stuff to deal with, but plasmid transformation is potentially something we'd want to include in our upcoming CIHR grant proposal, preferably with some preliminary data. The only major concern raised by the reviewers of our previous submission was that we didn't propose to evaluate the effects of point mutation changes in competence genes, just of deletions of the whole genes. The RA has developed a very nice way to make point mutations in genes cloned in E. coli, but we don't have an efficient way to transfer them into the H. influenzae chromosome. Well, we can transfer them into the chromosome by transforming competent H. influenzae cells with the mutated DNA, but this is only efficient if the DNA fragment is quite long (see this post), and finding the desired transformants requires laborious screening because there's no associated selectable marker. We're in the process of making a suitably long fragment of an interesting mutation she's generated, and I hope to be able to test this in the next week or two.
But the post-doc pointed out that we should also consider ways of testing point-mutation changes borne on plasmids, because these can easily be selected for. The strategy would be to test cells containing the mutant plasmid and a deletion that removes the chromosomal copy of the gene. One complication is that the plasmid-borne competence gene must have a promoter to drive its expression - ideally this will be its own competence-regulated CRP-S promoter. I'll have to check whether the short cloned fragment containing the RA's interesting mutation includes its promoter - if not, the longer versions will.
In most cases we'd be working with a gene that is essential for natural competence, so there are two ways to construct these strains. The first is to start with the strain carrying the chromosomal deletion and introduce the plasmid by electroporation. The RA has found that electroporation sometimes works very well and sometimes doesn't work at all - I don't know if she's ruled out the effects of the restriction enzymes in the H. influenzae cytoplasm, which are expected to cut up DNAs containing unmodified HindIII and HincII sites. The second way is to start with competent wildtype cells, introduce the plasmid by plasmid transformation with selection for the plasmid vector's antibiotic resistance, and then introduce the deletion by normal chromosomal transformation with selection for the inserted spectinomycin-resistance cassette.
I think this work needs a lot more thought, and soon, because the grant proposal deadline is Sept. 15. But last night I did a test of the basic plasmid-transformation method, using several DNA preps of plasmids based on the chloramphenicol-resistant shuttle vector pSU20 (two very old, and one new but of plasmid grown in E. coli). I transformed them into both competent H. influenzae and competent E. coli (as a control). I'll get the E. coli results today, but will have to wait till tomorrow for the H. influenzae results because the colonies grow very slowly on chloramphenicol plates.
How can you trust non-gardeners?
12 hours ago in The Phytophactor