The RA and I are working on a better method to isolate cells carrying 'unmarked' knockouts of the genes in the CRP-S regulon. She's made many mutations whose deletions are marked by addition of a SpcR StrS cassette. In principle these cassettes should be easily removable by (i) induction of a plasmid encoding an 'excisionase' of some sort that removes the cassette (details not important here) followed by (ii) selection for the resistance to streptomycin created by removal of the dominant StrS allele. But the plasmid is quite toxic and the selection doesn't work in our strain. We really want the unmarked mutations because many of the CRP-S genes are in operons where marked mutations in 'upstream' genes are likely to interfere with expression of genes downstream in their operon (to be 'polar' on them).
One possibility would be to make the marked mutation in the other strain (where the StrR selection works), and then transfer it by transformation into our strain. But we'd still have to screen our cells for the mutation, as there's no way to select for it.
The strategy we're testing now is to remove the cassette from the cloned marked mutations in E.coli, where everything works well, and to transform our strain with the unmarked fragment (from the plasmid or as a PCR product) and then screen the transformants for the expected phenotype (loss of transformation) or genotype (diagnostic PCR fragment). If the transformation occurs at a sufficiently high frequency we'll be set.
So we're doing a test. The RA amplified up two fragments, marked and unmarked versions of a rec-2 deletion mutation. I've transformed these into wildtype cells and will now screen these for loss of transformation; because she's already made the corresponding H. influenzae strains I already know that the mutants will not transform at all. Because the transformation may be inefficient I also included a small amount of NovR chromosomal DNA in the transformations and selected for acquisition of novobiocin resistance. I meant to also select the marked transformants for SpcR, but used 10-fold too little spectinomycin in my plates. (I'm going to reselect the cells to check this and get some known mutants.)
The big question is how I will screen the transformant colonies for loss of rec-2 function. The old way was to replica plate the colonies onto an sBHI agar plate that had been spread with antibiotic-resistance DNA, let the cells grow up overnight, and replica-plate the resulting colonies onto sBHI agar with the antibiotic. But this is far from quantitative, and the replica plating velvets we have always make ugly smears. A cleaner way, though more work, would be to pick the colonies into sBHI broth containing DNA (and cAMP?), and later spot the cells onto selective sBHI agar.
I think I should let the screening wait until tomorrow, and first do the Spc plating to find out what frequency of transformants I should expect.
End of summer &
8 hours ago in The Phytophactor