In addition to my GFAJ-1 arsenic experiments, I'm also doing work on our real research program, natural transformation in Haemophilus influenzae. We need to (yet again) revise our DNA uptake grant proposal for the Sept. 15 deadline, and I'm doing some work that will let us strengthen that proposal.
The previous version of the grant included a substantial section on making marked and unmarked knock-out mutations of all the competence genes, and one of the reviewers sensibly asked if we could also make point-mutation changes to these genes. This would indeed be very valuable, so we're trying to generate evidence that we can do this.
The biggest obstacle is not making the mutant versions of the genes (the RA can do that easily in E. coli), but identifying the rare H. influenzae cells that have integrated them into their chromosomes. We'd like to simply transform the mutated DNA fragments into competent cells, but this works very inefficiently when the DNA fragments are short. Unfortunately, short fragments work best for the mutagenesis steps.
My latest experiment tested the effect of fragment length on transformation efficiency I used a cloned DNA fragment containing the novR (novobiocin resistance) allele of the gyrB gene. This is a point mutation, in the middle of a 9.3 kb fragment. I did three test transformations, each using the same amount of the plasmid. Test 1 used plasmid DNA cut to release the 9.3 kb fragment intact. This gave a transformation frequency of 7 x 10^-3, about 10-fold higher than the control transformation using chromosomal DNA from a novR strain. Cutting the plasmid with restriction enzymes that gave a 4.8 kb novR fragment reduced the transformation frequency only slightly, to 4 x 10^-3, but cutting with enzymes that gave a 2.6 kb fragment reduced it 10,000-fold, to 4 x 10^-7.
Several factors complicate this experiment. I don't know the plasmid DNA concentration; I think it was about 100 ng/ml, which would be less than saturating. The plasmid insert contains several uptake sequences, but I don't know their distribution (I could dig this info out). The digest that gave the 4.8 kb fragment may not have been complete, in which case the true transformation frequency might be lower. The control transformation frequency was lower than I expected
We can conclude that our mutagenesis experiments should use fragments that are at least 5 kb long. If this gives a transformation frequency of only 4 x 10^-3 we'll have to do lots of screening (possible but tiresome), but the transformation frequency can probably be increased ten-fold by using more-competent cells and more DNA.
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