Constructing an Actinobacillus pleuropneumoniae antitoxin gene knockout: At the last report, I had what I thought were four independent knockout mutants, but my attempts to PCR- amplify the genomic segment containing the knockout were not working.
I eventually switched to using a different thermostable polymerase (NEB's standard OneTaq) rather then the fancier Q5 polymerase I had been using. Eureka - the PCRs all worked perfectly, giving strong bands of approximately the expected sizes.
...then I let everything sit around for a month while I dealt with other things...
Now I'm finally following up. The first step is to digest these PCR products with a few other enzymes that should cut in either the genomic segments or the inserted SpecR cassette. I've made rough predictions of the expected fragment sizes, which are all different for the ∆A mutant, wildtype cells, and the two mutants made by the Honours student (∆T and ∆TA).
The next step will be to do more PCR amplifications. My original amplifications used the F and R primers that amplify a 2.6 kb segment containing the toxin and antitoxin genes (~300 bp each). Now I'll use the F primer with the S-R reverse primer for the SpecR canssette, and the R primer with the S-F forward primer for the cassette.
If these both give the expected fragments then I'll (probably) send the PCR amplicons for each mutant to be sequenced.
If the sequencing confirms that the knocked-out genes are gone but the remaining gene is intact, then I'll give a sigh of relief.
Determining the competence phenotype of the Actinobacillus pleuropneumoniae antitoxin gene knockout: My first test of the transformability of my first two ∆antitoxin mutants showed transformation defects, but in later tests they transformed within the range of the wildtype control. But there was a lot of experiment-to-experiment variation in transformation levels (see graph below), so I'd like to do it one more time, to get clean publishable data.
Bonus DNA uptake results: Just before Christmas the grad student finished his DNA preps of H. influenzae chromosomal DNA fragments that had been recovered after being taken up into the periplasm of competent H. influenzae. He sent these to the former post-doc for sequencing, and the post-doc has now sent us some lovely preliminary results.
The grad student had used DNA preps that had been sheared to two different size ranges. We expected the genome coverage of the long fragments (mean length ~6 kb) to be fairly uniform, since almost all of them should contain at least one instance of the preferred uptake sequence motif. These 'USS' motifs are distributed fairly evenly around the chromosome, with a mean spacing of about 1 kb. We do see this, but with enough anomalies to keep things interesting. And we expected coverage by the short fragments (mean length ~0.25 kb) to be much more strongly dependent on chromosomal position, since many such fragments would not include a USS. And we do see this, again with interesting anomalies.