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 colonies descended from independent transformations, but for this experiment that's not important.

I think I'll try this today, using a normal log-phase culture rather that the EMS-mutagenized cells we'd use in the planned mutant-hunt:

1. Start with a mixture of competent cells and log-phase cells, such that I expect a transformation frequency of about 10^-7.  This frequency is higher than we would see in the planned mutant-hunt, because I want to have a predictable and easily measured number of transformants to start with.  
2. Incubate 5 ml cells at a density of 10^8 (OD600 = 0.03) with 1 µg MAP7 DNA for 15 min,  Plate an aliquot with and without novobiocin.  Dlilute the rest of the culture 100-fold with fresh medium and let grow for 1 hr, and then add novobiocin at 2 µg/ml to prevent further growth of NovS cells.
3. Plate aliquots again and at hourly intervals to check on the growth and survival of the NovS and NovR cells.
4. Just in case the dying NovS cells are toxic in the liquid culture, though they'll be much more dilute than on plates, after a couple of hours of novobiocin selection I'll wash the culture by filtration and resuspend the cells in fresh novobiocin medium and let them continue growing.  This will only take a few minutes and will also remove the residual NovR DNA.
5. After five hours of selection the NovR cells will have doubled about 10 times.  If the NovS cells survived growth in novobiocin but didn't divide I should see a 1000-fold increase in the frequency of NovR colonies on my plates, from about 10^-7 to about 10^-3.  If most or all of the NovS cells died the increase will be even greater.

If this works as expected, we'll be able to start our big experiment with a very large mutagenized culture and pool many more independent transformants.  This will give us a much more diverse pool of hypercompetent mutants for our sequencing.

 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

OK, the first try didn't work because I appear to have added bacteria to the sBHI agar instead of novobiocin (!!!) so what should have been novobiocin plates were plates with about 10^4 tiny colonies of H. influenzae embedded in the agar!

But the second try worked very nicely.  The red lines are the planned experiment, with a small number of competent cells added to a 100-fold excess of non-competent cells; the green lines are just the competent cells by themselves.  Each cell prep was incubated with NovR transforming DNA for 15 minutes, and then novobiocin was immediately added to the culture.  The non-competent cells (open red squares) continued growing a bit and then stalled (the competent cells had already stopped growing because they had been starved to induce competence).  After two hours the transformed NovR cells (solid red and green squares) started doubling and the non-transformed NovS cells (open red and green squares) started dying, just as they should.  By the next morning the cultures were thick and consisted of only NovR cells.

This result means that we can incorporate selection for NovR transformants in broth in the mutagenesis experiment and so won't  have to distribute the transformation mixture over hundreds of Nov plates.