1. I made lysates of all four phages. Making lysates of HP1 phage is an act of faith, because inducing the prophage doesn't cause most of the cells in the culture to lyse. When prophage lambda is induced in a lysogenic E. coli culture, the culture density increased for an hour or so and then the cells all lyse (the culture goes clear, with bits of threadlike cell debris visible). But the induced HP1 lysogens just continue to get more dense, and never really appear lysed (though sometimes some debris is visible).
I've only titered the wildtype lysate because the 34°C and 41°C incubators can't be moved to our lab until next week. Despite the lack of obvious lysis, the lysate contains about 5 x 10^10 plaque-forming units per ml, which is just fine. If the temperature-sensitive mutants behaved similarly I'll have lots of phage for my recombination assays (the titer of the phage needs to be about ten times that of the cells, to get the necessary multiplicity of infection).
2. I've finished transformation assays on three of our new competence-gene knockouts. Two of the genes, comA and comC are in the big comABCDEF operon; both mutations are 'unmarked' deletions and not expected to interfere with expression of the downstream genes in the operon. The RA tells me that all of the genes in the homologous Neisseria meningitidis operon (pilMNOPQ) are needed for functioning of the pilQ-encoded secretin pore through which DNA is taken up (I haven't looked at the paper(s) yet), so we expect knockouts of these genes to completely eliminate transformation. However, although the comC knockout did give no transformants at all, the comA knockout had a normal (wildtype) transformation frequency. I think this probably isn't just a mistake on my part (e.g. a strain mixup), because the undergrad who worked on these mutants also saw high transformation frequencies for both the marked and unmarked comA mutants. But we'll need to use PCR to confirm that the cells I'm using do have the right deletion.
I made six more mutants competent yesterday. I'll analyze the results today, once the colonies get big enough to count. And I'll streak out more mutants to transform tomorrow (I'm on a roll).
I also froze 3 ml of competent cells from each prep, to use in the phage-recombination assays. These cells will also be used for the PCR-checking of genotypes. The RA will do this - I need to ask her about how much preparative work is needed - can she just use the frozen cells directly in PCR or do we need to do DNA preps first?
Friday Fabulous Flower - Squashes & pumpkins
2 hours ago in The Phytophactor