I want to create a pool of cells with random point mutations in the H. influenzae murE gene, and to select and screen this pool of cells for hypercompetent mutants. I'm going to do this by mutagenizing the DNA with the chemical mutagen ethyl methanesulfonate (EMS) in vitro and then transforming it into cells, rather than mutagenizing cells.
One unanticipated benefit of the in vitro method is that the mutation spectrum is better. With in vivo mutagenesis, EMS produces mainly GC-to-AT transition mutations by alkylating guanines in DNA, creating O-6 ethylguanine which mispairs with T instead of C during DNA replication. (info from Wikipedia). But the in vitro work found a much less biased distribution, with 42% GC-to-AT transitions, 34% AT-to-GC transitions, and 24% GC-toCG transversions.
Step 1. Cut chromosomal DNA of strain RR
Here's the map:
This pre-digestion step could probably be omitted if necessary, because random fragmentation of the DNA will accomplish almost as much. But it shouldn't hurt, and it might double the frequency of cotransformation. But I just looked at some old cotransformation data, and I see 60-70% linkage (selecting for CmR gives the linked murE allele), which is very good
Step 2. Soak this DNA in an EMS solution for 1 hr.
Step 3. Wash the DNA and transform it into competent wildtype cells. Use about 100 ng DNA per ml, so that each cell is likely to recombine only a single DNA fragment. As a control, transform the same cells with DNA from the chloramphenicol-resistant murE749 strain.
Step 4. Select for chloramphenicol resistance, to enrich for cells that have recombined in murE. This will also confirm that the level of DNA damage was not so high as to limit transformation. I should be able to get many thousands of independent transformants.
Step 5. Pool chloramphenicol resistant colonies, creating separate pools from independent sets of transformants. Aim for about 5 pools. Freeze some of the cells of each pool. Make a pool for the control transformants too.
How many colonies should be in each pool? I want enough colonies per pool that each is likely to contain at least one hypercompetent mutant - how many colonies will this be? I know of three mutations that produce hypercompetence, which would let me predict the minimum expected frequency of hypercompetent colonies if I knew the frequency of mutations in the DNA and the degree of linkage in the transformation. I can measure linkage by doing colony assays on the control transformation. The enrichment can increase the frequency of hypercompetence by 1000-fold, if all the mutants are as hypercompetent as the ones we have. So if the frequency of hypercompetence in the chloramphenicol-resistant transformants is 1/1000, I should put at least 1000 colonies in each pool. If it's less, I should put more.
Step 6. Grow the pooled cells in sBHI at low density for a few hours, then transform with cloned or PCR'd NovR DNA (or a different marker?). Plate on nov plates. Do this with the control murE749 transfornation too.
Step 7. Screen individual NovR colonies for hypercompetence by touching them to nov plates and then resuspending the rest of the cells in sBHI containing MAP7 DNA and plating on Kan (or Nov?) plates. Do only 10 colonies per pool, or 1/1000 as many colonies as went into the pool? I expect most of the control colonies to be hypercompetent.
Step 8. For each pool, pick one or two high-transformation colonies from their toothpicked plate, and retest their competence with a simple time course.
Step 9. PCR and sequence the murE genes from5 or 10 of the confirmed hypercompetent mutants (depending on how many I get, of course). Are the known mutations present? New mutations?
First we should test different levels of mutagenesis:
The protocol we have (Lai et al.) says to use 1 µg DNA in 20 µl 10 mM EMS for 1 hr; this gave 5-6 mutations per kb in the clones they sequenced. It also reduced the transformation efficiency of the plasmid insert they mutagenized to about 60%. If they carefully standardized the amounts of DNA, this reduction should have been a direct consequence of DNA damage and repair processes, since they were not selecting for function of their mutagenized insert.
5-6 mutations per kb sounds pretty good for us (but see next post, which suggests we want fewer), since about half of them will be silent, but I think we should first try a wide range of concentrations. For the cell mutagenesis (many years ago) I used 50 mM for 45 min and 80 mM for 30 min (RR expt # 181), but we want much heavier mutagenesis here. So here let's try 0, 2, 5, 10, 20, 50, and 100 mM - that's 7 DNA samples to do transformations with.
Two assays for the extent of mutagenesis:
1. (To identify an optimal concentration) Mutations creating low-level resistance to novobiocin: Mutagenize any novS DNA (e.g. RR805) and transform into KW20 and select for low-level novobiocin resistance (1 µg/ml rather than 2.5), to check the efficacy of the mutagenesis. There should be an optimal dose of EMS, above which the frequency of nov resistance drops because the DNA is too damaged to recombine or contains too many mutations that block gene function.
2. (To identify concentrations that are too high) Mutations that inactivate the CAT cassette: Mutagenize RR805 DNA and transform KW20 to chloramphenicol resistance. At some EMS dose the transformation frequency will decrease because the DNA is too damaged to recombine or contains too many mutations that block gene function. (This test could also be done with any point mutation creating antibiotic resistance.)