I want to take advantage of having selectable alleles linked to each of the genes where mutations produce hypercompetence. That's 1) the StrR point mutation linked to sxy (about 50 kb; 50% cotransduction), 2) the CatR cassette linked to murE (about 4 kb; 90% cotransduction?), and the CatR cassette tightly linked to rpoD (~100 bp; > 90% cotransduction).
Last time I tried to do this by doing the EMS-mutagenesis in vitro (See blog posts: here and here). Call this strategy C. I directly mixed EMS with DNA of three strains carrying the above selectable markers and wildtype alleles of the hypercompetence genes, and then transformed the mutagenized DNAs into competent wildtype cells, selecting for each of the marker strR and CatR alleles. But this failed. I didn't get any low-level novobiocin resistance mutations that would have indicted that the EMS caused mutations, and I didn't get any hypercompetent mutants. I suspect that transforming cells with EMS-damaged DNA is a very inefficient way to create mutations.
This time I'm going back to doing the mutagenesis in vivo (call this strategy B), incubating the three marked strains with EMS and allowing them to grow for 1-2 hr to convert the damage into mutations. Then I'll isolate the DNA from each mutagenized culture, and use this to transform wildtype cells to the three markers. This will give me pools of cells that have experienced a high mutation rate in the neighbourhood of sxy, murE and rpoD.
But before I do this I need to do the math, to see if this is really any better than just mutagenizing wildtype cells and screening them all for hypercompetence (call this strategy A). Having the linked markers will certainly be handy later, once I've found hypercompetence mutants and want to find out which gene they're in. But will using them as described above really let me find more mutations in these specific genes?
OK, I've laid out a situation with realistic numbers, and I've run it by the PhD student and the summer student. Bottom line: Strategy B does not enrich for mutations in the desired region. It's only strength is that it allows use of much higher concentrations of EMS than would be tolerated in Strategy A.
Here's the numbers analysis for strategy A:
- Start with 10^9 cells. (Below I'll consider whether fewer would be OK.)
- Treat with EMS (0.08M for 30 min). Previous work suggests that this creates about 1 mutation per surviving cell.
- Grow cells for about 3 hr or more, keeping cell density below OD600= 0.2. The goal is to allow enough time for the cells to recover from the DNA damage, undergo two rounds of DNA replication to convert some damage into G->A mutations, and express the mutant hypercompetent phenotype. Assume that the cell numbers increase 10-fiold during this period.
- How many mutations will we have? The genome has about 4 x 10^5 Gs. Mutations at some of these will be lethal or sub-lethal, so assume about 2 x 10^5 positions where mutants have normal or near-normal growth. With 10^10 cells, we will have about 5 x 10^4 occurrences of each mutation, on average.
- How many hypercompetence mtuations will we have? 10 of these are positions we have already found to be sites of hypercompetence mutations, so in our 10^10 cells we'll have at elast 5 x 10^5 hypercompetent cells.
- To select for these hypercompetent cells, transform the OD600=0.2 cell population with the 8 kb NovR DNA fragment from plasmid pRRnov1. This DNA transforms much better than the equivalent PCR fragment or than chromosomal DNA with the equivalent mutation.
- The normal cells will transform at a frequency of about 10^-8 - 10^-7. (I'm guessing here; with chromosomal DNA it's ~10^-9.) That would give about 100-1000 novR colonies from the 10^10 cells.
- The hypercompetent cells will transform at higher frequencies: rpoD: cells with the known rpoD mutations will probably have a TF of 10^-4 - 10^-3, so the 5 x 10^4 cells of each mutation would give about 5-50 novR colonies. sxy: cells with the known sxy mutations will probably have a TF of 10^-3 - 10^-2, so the 5 x 10^4 cells of each mutation would give about 50-500 novR colonies. murE: cells with the known murE mutations will probably have a TF of 10^-2 - 10^-1, so the 5 x 10^4 cells of each mutation would give about 500-5000 novR colonies.
- According to this analysis, most of the colonies will be hypercompetent mutantx, and most of the hypercompetence mutants we find will probably have their mutations in murE. Of coulrse this only considers the hypercompetence mutations we already know about.
Things to do first:
- Check that our EMS stock is still good, by mutagenizing wildtype cells and scoring low-level novobiocin resistance. A resistance frequency of ~5 x 10^-6 is approximately one mutation per viable cell.
- Make a big prep of pRRnov1. The yield of this plasmid is often poor so this may take several attempts and large volumes. Check the transformation frequencies and efficiencies this DNA gives.