Mutagenesis results

I don't have any novobiocin-resistant transformant-mutants after 24 hr (though slow-growing colonies might appear later), so I can't use that to tell how effective the mutagenesis was.  But I have tons of chloramphanicol-resistant ones at the low exposures to EMS (2, 5 and 13 minutes), 100-fold less at 30 minutes exposure and none at 60 minutes exposure (the highest dose).  This tells me that the EMS was doing its job, and that the DNA damage caused many potential transformants to have lethal mutations either in the CAT cassette or in nearby genes in the recombination tract.

So I think I'll go ahead and make pools of colonies from the 5-min and 13-min treatments and enrich them for hypercompetent mutants by selecting for StrR transformants in log-phase cultures. Then tomorrow I can screen these for hypercompetence by our crude colony-transformation assay.

Why not also the 2-min treatment?  OK, I'll include one pool of those too.

I'll have four five pools (10^4 and 10^5 transformants from each of the two treatments), which will be easy to handle.  What control cultures should I include?  RR805 (murE+) will give negative control colonies, and RR797 (murE749) will give positive control colonies.



* One reason to not use the ~1000 CmR colonies from the 30-min dose is that these are less likely to have recombination tracts extending all the way from the CAT cassette to murE.  That's because this segment contains two essential genes (ftsI & ftsL), and recombination tracts that cover the CAT-murE distance are much more likely to have had a lethal mutation in one of these genes than are tracts that don't reach to murE.