One of the talks at the Analytical Genetics conference was about advances in 'recombineering'. This is a way of dramatically increasing the homologous recombination rate in E. coli by expressing recombination-promoting genes of phage lambda This enhanced recombination is combined with introduction (by electroporation) of DNA fragments containing site-directed mutations (made with PCR), to give a very efficient method of changing chromosomal alleles.
The new work applies this to single-stranded DNA fragments synthesized as oligonucleotides. When the electroporated DNA is single stranded it's possible to eliminate two of the usual three lambda proteins (the two that are toxic to the cell), instead using only the non-toxic Beta protein. Now that the toxic proteins aren't needed the Beta protein can be constitutively expressed from a plasmid without reducing cell viability.
There were also some very interesting discoveries about how mismatch repair acts, and how it can be circumvented by incorporating adjacent mutations to the desired one, but I'll have to post about these later because I don't have my notes handy. For some of the ssDNAs used the transformation frequencies were greater than 10%, so transformants could be identified by simple screening.
Anyway, it occurred to me that this might be useful for enhancing transformational recombination in E. coli and maybe in H. influenzae too. So I'll be getting the beta-expressing plasmid from its creator and introducing it into various E. coli strains. We'll then test whether these strains can be transformed with chromosomal DNA as H. influenzae can. We might also test whether using a synthetic oligo works, as it does by electroporation.
The other thing we could do is transfer the Beta insert to a plasmid that can replicate in H. influenzae (if the one it's in can't). Then we could see how Beta affects transformational recombination. If transformation dramatically increases, as it might, we could offer the plasmid to people working on other Pasteurellaceae species who are trying to get transformation working (or working better) in their species. We could even add this transformation to our 'recombinome' project, asking how Beta-catalyzed recombination differs from Rec-1-catalyzed recombination.
If at first you don't succeed, put it all back they way it was and keep lumbering along
1 hour ago in The Phytophactor