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:

1. Wildtype H. influenzae Rd
2. 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.
3. 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 for expression of the murE-mutant hypercompetence phenotype?  Hard to say, since we don't know how the mutations cause their phenotype.  Ideally I would do an experiment to find this out.  (How? Not easily, it seems...   I could transform competent wildtype cells with DNA from a CmR murE749 mutant, selecting for CmR after 1 hr expression time, and then at intervals (every hour?)  transforming aliquots of the culture to NovR with MAP7 DNA.  But the cells would initially have to be competent, so I'd have to grow the culture for at least a couple of hours at low density to grow-out this competence before I would be able to detect any log-phase competence caused by the murE mutation...)  Instead I think I'll just hope that 2 hr of log-phase growth is enough time for a new murE mutation (or any other mutation) to cause hypercompetence.  Wait, is there any compelling reason not to grow them longer?  The volume will keep getting larger with the repeated dilutions, but I could just freeze some of the cells after 1 or 2 hr and continue growing the rest for another hour or two.  The frequency of the mutants I'm looking for shouldn't change with growth, since none of the hypercompetence mutations we know of slow growth.

So new plan:  Grow the mutagenized cultures for 90 minutes.  The OD should be back up to about 0.1.  Freeze 3/4 of the cells (concentrate by filtration before freezing).  Dilute the rest back to OD 0.025, grow for another hour (OD back to 0.1), and then add transforming DNA.  

The postdoc is making me a prep of NovR PCR fragment that I can use for these transformations.  I'll want to do a test transformation first, using this DNA with normal competent cells, to determine how much DNA to use.  We don't want to use MAP7 chromosomal DNA because (1) the efficiency is low because most of the DNA is from other parts of the chromosome, and (2) it will transform other parts of the competent cells' chromosomes, potentially removing the mutation we want to isolate.

Incubate the cells with DNA for 30 min, add DNaseI and wash the cells by filtration to remove both the DNA and dead cells.  Resuspend in medium with novobiocin and grow for at least 6 hr (or overnight) before plating on nov5 plates. (We usually use nov at 2.5 µg/ml, but NovR transformants grow just as well on plates with 5 µg/ml, and most spontaneous NovR mutants don't.)  Freeze some of the cells instead of plating them all.

If the cultures are grown overnight with novobiocin,  I should probably increase the novobiocin to 5 µg/ml after the first few hours.  In the morning I could plate some of the cells for NovR colonies, and just grow some in log phase for 2-3 hr before transforming them with KanR DNA to select the ones that are genetically hypercompetent (eliminating the ones that were accidentally competent for the first selection).  Alternatively I can wait and pool the NovR colonies that grow up on the plates.  Because the culture should be already enriched for hypercompetent mutants I won't need to worry about 'bald spot' effects but can plate relatively dilute cultures on the kan plates.  (I won't assume this but check by plating different concentrations of course.)

Next I pool all the transformants from each StrR or CmR culture (KanR if I've done the second round of selection, NovR if I haven't) and prep DNA from them.  I use the DNA to transform competent wildtype cells to StrR or CmR, grow the pooled cells into log phase, and select for hypercompetent cells with NovR DNA.  (This could be a PCR fragment or chromosomal DNA (wait, do I have a NalR strain in the freezer?).)

For the wildtype culture, I can just do one more round of hypercompetence selection, with NalR, or I can do an unselected transformation (or select for NovR as an unlinked marker) and then do the NalR hypercompetence selection.

Pool the NalR transformants from each culture, extract DNA and sequence.  If there are hypercompetent mutants we expect to see peaks of novel alleles at and around the sites of the mutations.

So, the whole experiment:  First streak out the cells.  Then dilute and grow, mutagenize, wash, dilute and grow, freeze, dilute and grow, transform with NovR, wash, grow with nov, freeze, grow with nov, plate.  (Pool), dilute and grow, transform with KanR, plate.  Pool KanR, make DNA, transform to CmR or StrR, plate.  Pool, dilute and grow, transform to NalR, plate, pool, make DNA, sequence.

AAACCKKK!  Maybe it will be clearer if I partition it into days?

Day -1: Streak out the cells.  (3 strains)

Day 0:  Inoculate single colonies overnight.  (3 strains)

Day 1: Dilute and grow, mutagenize, wash, dilute and grow, 

            freeze, dilute and grow, transform with NovR, wash, 

            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)

Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))

Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)

Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)

Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)

Well, at least I now know what I'll be getting into.