Today at lab meeting we discussed our plans for creating specific point mutations in competence genes. This is something that one of the reviewers of our CIHR proposal wanted to see, and we think we can do it before the next grant deadline (Sept. 15).
The RA has a clever way to make any desired point mutation in our cloned competence genes, and we can easily introduce such mutations into the H. influenzae chromosome by natural transformation. The big problem is getting a high enough transformation frequency that we can identify the desired mutants by PCR. Because the mutations don't have an associated antibiotic resistance we won't be able to select for them, and because we won't usually know the effect of the mutation in advance we may not be able to screen for an expected phenotype.
We know that transformation frequency depends on the length of the fragment, the kind of heterology (single nt, insertion/deletion, etc), and the presence of uptake sequences.
We want to do some preliminary experiments to check the effect of fragment length. In the past I had gotten transformation frequencies of better than 5% with a 9 kb restriction fragment containing a cloned novobiocin-resistance point mutation, but more recent experiments with shorter fragments had given frequencies that were 100-fold to 1000-fold lower. So we were planning to do a new test, first cutting this novR fragment to different lengths with restriction enzymes and then measuring the effect on transformation frequency.
But while looking up the restriction map of this plasmid I discovered that I and my first technician had done a version of this experiment already, back when we were first working with this plasmid (RR#196). I think we weren't testing how fragment size affects transformation, but just finding out whether we should cut the insert free of the vector.
The plasmid has a 9.25 kb fragment of chromosomal DNA in a 2.3 kb pSU vector; the novR mutation is somewhere in the 2.4 kb gyrB gene (the postdoc knows where but he's not here right now).
The technician's transformation frequency results: Using an uncut plasmid: 0.024; linearizing the plasmid, leaving the vector attached to the insert: 0.061 and 0.086; cutting the insert free of the vector: 0.093; cutting the plasmid once, about 2 kb from one end of gyrB: 0.051; cutting the plasmid once, 70 bp from one end of gyrB: 0.036. (One time, I got a transformation frequency of 0.22 with this DNA!) Transformations that used the same novR marker carried in chromosomal DNA gave a transformation frequency of 0.011.
These are lovely high transformation frequencies - if we can get similar frequencies with our engineered mutants we'll have no trouble identifying them by PCR. Another limiting factor is how much DNA we can use. I don't have a good estimate of the DNA concentration the technician used in this experiment, but in a later experiment (RR#860) I used 100 ng of insert, from plasmid grown in either H. influenzae or E. coli, and got transformation frequencies of 0.045 and 0.07 respectively. So if the RA can generate 100 ng of mutant fragments (perhaps by long PCR) then we'll be all set.
But generating long fragments is a pain, so we still need to test the efficiencies of transformation with shorter novR fragments to find out how long her fragments need to be. This means that we need to grow up a prep of the plasmid. I've had problems with yield of this plasmid in the past (it's a low-copy vector), but the post-doc is confident he can get lots, so I'm streaking the strain out for him now. If yields suck we can always reclone the insert in a high-copy vector.
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