A better strategy for finding new hypercompetence mutations in murE

If we did decide to buy some EMS (it's cheap and readily available from Sigma) and repeat the mutagenesis, I think we should use a different strategy to select for hypercometent mutants.  Specifically, we should focus on getting more  strains whose hypercompetence is caused by mutations in murE (see this old post for a description of the murE results).  We have four such mutants now, which change two different amino acids, and we don't understand how they work.  Having more independent mutants would help clarify the situation, whether we get new mutations or just more of the same mutants.

How to do this?  We would start by mutagenizing cells with EMS, but instead of using wildtype cells we'd use cells carrying a neutral chloramphenicol-resistance insertion (CmR) that's within a few kb of murE.  The mutagenized cells would be incubated in log phase with a NovR DNA fragment, and the rare transformants wuld be selected by plating on novobiocin and pooled.  Since the transformation frequency of wildtype cells in log phase is less than 10^-9, many of these should be hypercompetent mutants.  DNA from the pooled transformants would be used to transform wildtype cells, selecting for CmR to enrich for mutations in and near murE, and these 'backcrossed' transformants would again be selected for hypercompetence by transformation in log phase, this time using MAP7 DNA and selecting for streptomycin resistance.  DNA from the pooled transformants would be sequenced, as would individual hypercompetent isolates.

We need to think about the numbers.  Say 1-5% of the original NovR transformants are hypercompetent strains with murE mutations (in the original experiment we found 4/150).  If we pool their DNA and backcross, selecting first for CmR, 1-5% of the colonies will have murE hypercompetence mutations.  Even if these mutants were only 100-fold more competent in log phase than normal cells, selecting for these by transformation to StrR would give mostly colonies with the desired mutations.

(The murE mutants we have are about 10^6-fold more competent, so we might miss mutations giving weaker hypercompetence phenotypes...)

Timing:

1. Day 1.:Streak out the CmR strain.
2. Day 2: Grow the CmR strain and mutagenize for 30 min with EMS.  Wash away the EMS, freeze some of the cells for later work. Dilute the rest right away and grow in log phase for 2-3 hr or more.  Add NovR DNA for 30 min and plate on lots of plates.  
3. Day 3: At this stage each hypercompetent NovR colony is likely to be an independent mutant.  Pool all of them and isolate DNA.
4. Additional Day 2?: We want many hundreds of transformants, and can scale up the mutagenesis and transformation cultures,  but the selection is limited by the need to not put more than ~5x10^7 NovS cells on each plate.  Maybe we should also try enriching for NovR before plating, by adding novobiocin to the broth and growing for a few hours or overnight.  This would let us screen a lot more cells and thus find more mutants, although we'd sacrifice independence.  Again we'd then isolate DNA from the NovR culture or pooled NovR colonies.
5. Still Day 3: Transform competent wildtype cells with this DNA, selecting for CmR.   The expected transformation frequency is about 10^-4 to 10^-3, so we can easily select many thousands of independent transformants to pool.  1-5% of these should have the desired hypercompetence mutations.
6. Day 4: Grow the pooled transformants and transform them in log phase to StrR.  
7. Day 5: Almost all the transformants should be the desired hypercompetence mutants. Pick some of these for competence testing and pool the rest.
8. Prep the DNA of a number of individual isolates and of a large pool of colonies.  Sequence these.  Look for specific mutations in the individual cultures and for enrichment of mutations in the pool.

Should we also do an unfocused search for any hypercompetence mutations?  We could do this in parallel, replacing the selection for CmR on Day 3 with selection for NovR.  We might still get mainly mutations in murE, because these give such a strong phenotype.  If we wanted to target mutations in sxy, we could first do a second round of selection for transformation using StrR MAP7 DNA and then select for StrR in the backcross.  Since StrR is not very close to sxy we'd want to be gentle with our Day 3 DNA prep so the fragments were long.