Selecting for rare NovR cells by enrichment in broth - would it work?

In the previous post about searching for new hypercompetent mutants I mentioned that our ability to find rare NovR transformants in a log-phase culture is limited by the need to put relatively small numbers of NovS cells (less than 5 x 10^7) on each plate.  If we use more cells we see large 'bald spots' where the NovR cells are unable to form colonies; we speculate that this is because of toxic effects of too many NovS cells dying around them.  When the problem is severe we see no NovR colonies at all even though hundreds were plated. One solution is just to distribute the cells over more and bigger plates.  Scaling up by ten-fold is easy, but scaling up by 100-fold is a lot more work and more expense for plates and medium.  In the experiment we're now considering, we expect the NovS cells to outnumber the NovR cells by about 10^8 to 10^9, so we'd like to scale up by a thousandfold. An alternative that we've never tried is to add novobiocin to the liquid culture for a few hours before plating the cells on agar medium containing novobiocin.  This scaling up would let us amplify the rare NovR cells by letting them double repeatedly while the NovS cells stalled or died.  Ten doublings (about 5 hr of growth) would bring the NovR density up from 10^-9 to 10^-6, so that plating on a moderate number of plates would capture the full diversity of the initial transformant population.  We would no longer be able to assume that separate NovR coloni...
Source: RRResearch - Category: Medical Scientists Authors: Source Type: blogs