Field of Science

Are the old mutagenized cells worth using?

Last month I wrote a post about an old experiment (What can I recover from an old failed experiment).  I concluded that some of the frozen mutagenized cells might be worth using, but that I would first need to test how effectively they had been mutagenized.  Now it's time to do that.

The cells were incubated with the mutagen ethane methylsulfonate (EMS), which alkylates G bases and causes G->A transition mutations.

I can test the efficiency of this mutagenesis by plating cells on novobiocin agar plates, using a lower-than-normal novobiocin concentration (1 µg/ml rather than 2.5 µg/ml).  I also need to test the overall viavbility of the frozen cells, by plating them on plain agar plates, but I'll need to do this anyway to test the mutagenesis. 

I have two tubes of each of six cultures (three strains at two EMS concentrations).  Each was grown for 90 min after the EMS treatment, to allow DNA replication to convert the DNA damage to mutations:
  • B and C: KW20 (wildtype), 0.05 mM and 0.8 mM EMS
  • D and E: RR514 (StrR, linked to sxy), 0.05 mM and 0.8 mM EMS
  • F and G: (RR805 (CmR, linked to murE), 0.05 mM and 0.8 mM EMS

I'll thaw and test one tube of B and one of C; these should be representative of the others.

Rather than just discarding the remaining cells I thawed, I could at the same time put these cells through the next step, by growing them at low density and transforming them with either novR chromosomal DNA or a novR PCR fragment (better because higher transformation efficiency).  But I won't do this, because first I need to find the novR PCR fragment and test it.

I just checked my old notes.  In expt #1290 I had tested the PCR products (novR and nalR) and found that they gave only slightly higher transformation frequencies than MAP7 chromosomal DNA.  This is surprising, since the effective concentration of the resistance-conferring fragments is much higher in the PCR DNA prep (maybe 10-50-fold higher, depending on the concentration of the PCR DNA, which wasn't measured.  The MAP7 DNA was used at a concentration that's saturating for transformation.

BUT, on more carefully re-reading my old notes (as usual not as limpidly clear as I would desire), I can't be sure that the cells I have saved are the cells I need (they might instead be cells that have already been incubated with the wrong DNA).  I think I'd better abandon this mess and start fresh.

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