By midday today I'll have checked my strains and made my DNA.
The strains are RR805, which has a CAT cassette linked to the murE+ gene (normal competence), and RR797, which has the same cassette linked to the murE749 hypercompetence allele and a StrR point mutation elsewhere in the chromosome. I've checked their antibiotic resistances, done platings that will confirm their competence phenotypes (will count colonies this morning), and made crude DNA preps (I'll complete purification this morning).
Next I should do the mutagenesis dose-response curve, and I've now realized that this experiment can also be used for the first hunt for more hypercompetence mutants.
Set up one tube containing 12 µg RR805 chromosomal DNA in 120 µl water or TE, at 37 °C.
Take a 20 µl time = 0 sample (see below).
Add EMS to the remaining DNA, to a final concentration of 50 mM.
Take samples at time = 2, 5, 12, 30 and 60 minutes. Immediately add each sample (including t = 0) to 100 µl of 5% sodium thiosulfate, which will inactivate the EMS and stop the mutagenesis.
The t = 2 sample will have had about 6-fold less exposure to EMS than used by the Lai et al. paper, and the final sample will have had 5-fold more.
Add NaCl to each sample to 0.15 M and add 2 volumes of ethanol to precipitate the DNA. Rinse the pellets (probably invisible) with 70% ethanol and air dry. Resuspend each in 50 µl TE. (If the invisibility of the pellets is a problem I could add some E. coli DNA as carrier, since this won't interfere with the subsequent transformations.)
Thaw out lots of vials of frozen competent KW20 cells (wildtype). I need one tube for each of the 6 DNA samples, and also one for RR797 DNA (chloramphenicol resistance control) and one for MAP7 DNA (transformation control).
Add 2.5 µl (= 100 ng) of each DNA to a tube containing 1 ml of cells. Incubate for 15 min at 37 °C.
Add 3 ml sBHI and incubate for 90 min longer, to allow expression of the chloramphenical resistance.
Dilute and plate on plain plates (10^-6, 10^-5), Nov1 plates (for low-level novobiocin resistance, plate undiluted and 10^-1) and Cm1 plates (plate 10^-3, 10^-2, 10^-1 and undiluted).
Freeze the remaining transformed cells in case I want to do more with them later.
Analysis and next steps (Friday):
Use the colony counts to assess the extent of mutagenesis and gene inactivation. For doses that gave high NovR mutagenesis without reducing the CmR transformation rate, make pools of the CmR colonies from plates that have >1000 colonies (one pool per plate).
Then I cna grow each pool to early log in sBHI and transform it with StrR DNA to enrich for hypercompetent mutant.
Then I'll screen individual StrR colonies for hypercompetence by mixing them with MAP7 DNA and plating on Nov.
What if I don't get any NovR mutants?
My previous use of EMS, mutagenizing cells, not DNA, gave NovR mutants at about 10^-6 of the survivors. If this was the level of NovR mutations in my mutagenized DNA, the transformation assay probably wouldn't detect their presence because only about one cell in 1000 will have recombined the nov-containing DNA fragments, giving a transformation rate of 10^-9, below the detection limit. But I expect the mutation rate to be much higher for the pure DNA, so I'm hoping that I'll see significant increases in resistant colonies.
If I don't? I could just go ahead and screen a couple of the high-dose CmR pools for hypercompetent mutants anyway, since if I find some then I can just forget about the Nov test. If I don't find any hyprecompetent mutants I should repeat the mutagenesis using a NovS DNA fragment as control.
Friday Fabulous Flower - tropical springiness
1 hour ago in The Phytophactor