For at least half of our mutants we have independent evidence of the mutant's phenotype (a previously reported knockout mutant or a second mutant that we made). Some of the remaining mutations don't cause any detectable phenotype, so they don't need to be tested. There are only three that really deserve testing, comN, comP and comQ. But rather than constructing plasmids that carry and express each gene, I'm testing them by backcrossing. That is, I'm making DNA from the 'marked' SpcR version of each mutation and using this DNA to transform wildtype cells to SpcR. This will select for cells that have acquired the knockout mutation. The transforming DNA fragment is likely to have introduced short segments of flanking chromosomal DNA, but this will be less than 1% of the genome. I'll then test the transformants to see if they have acquired the expected competence defect. If so we can be reasonably confident that the mutation we created is the cause of the defect.
I've purified the DNA, transformed it into wildtype cells, and streaked the transformants onto Spc plates. (5 µl of a 1/00 dilution of a crude DNA prep from 2 ml of cells gave more than 10^6 transformants!). Tomorrow I'll make the cells competent and transform them. If I don't get any transformants then we just need to make a few revisions to the text of our manuscript and resubmit it.
Once I have the mutant plasmid (checked by restriction digest; it should have the same structure as the one my previous plan would have generated) I'll just cut out the insert and transform it into wildtype H. influenzae, selecting for SpcR. If the double mutant is competent, I'll know that HI0659's job is to prevent HO0660 from doing something that blocks competence. If not, not.