The EMS mutagenesis mutant hunt - progress and plans

I wrote about this general experimental plan here, and described the preliminary test of the NovR selection here.  The EMS mutagen has been ordered and should arrive within the next few days.  The experiment isn't as urgent as we originally thought, because the DNA sequencing plans have changed, but it still needs to be done soon, so I'd better be ready to get started.  Here's the plan: Do the initial mutagenesis in three different strains: Wildtype H. influenzae Rd H. influenzae Rd carrying the normal sxy gene and a streptomycin-resistance mutation.  StrR is only about 50 kb from sxy, so we can use it to enrich for hypercompetence mutations in sxy. H. influenzae Rd carrying the normal murE gene and a chloramphenicol-resistance mutation about 4 kb from murE, which we can use it enrich for hypercompetence mutations in murE. To minimize spontaneous competence the cells will be pre-grown in sBHI at low density for at least 2 hr. Each strain will be incubated with 0.0, 0.05 and 0.08 mM EMS for 30 min at an initial OD600 of 0.05. Maybe the volume will be 10 ml. After the 30 min mutagenesis each culture will be thoroughly washed by filtration to remove the EMS and resuspended in fresh sBHI (50 ml? 100 ml?).  First plan: The cultures will be grown for 2 hr - that should be enough for any mutational changes in sxy to cause elevated competence.  The density will need to be kept low - ideally below OD600 = 0.1.  Will it be enough time...
Source: RRResearch - Category: Medical Scientists Authors: Source Type: blogs