Field of Science

What can I recover from an old failed experiment

About 18 months ago I did a big mutagenesis experiment, intending to isolate new hypercompetent mutations.  I made several mistakes and the experiment was a failure, but I did freeze stocks of intermediate cultures.   At the time I thought that some of these could be used in a future attempt, because they came from stages before the mistakes were made.

I still want to repeat this experiment, and I just found the stocks in the -80 °C freezer.  Now I need to decide which are potentially useful, and throw out the rest.  Here's photos of what I found:


The letters A-G refer to different strains, each with a wildtype version of a gene known to give rise to hypercompetence-causing mutations, and to different levels of mutagenesis
  • A, B & C: wildtype cells, incubated in 0 (A), 0.05 (B) and 0.08 (C) M solutions of the mutagen EMS.
  • D & E: strain RR514, which has a Streptomycin-resistance mutation (StrR) close to the wildtype sxy gene, incubated in 0.05 (D) and 0.08 (E) M solutions of EMS.
  • F & G: strain RR805, which has a chloramphenicol cassette (CmR) inserted within a few kb of (= closely linked to) the wildtype murE gene, incubated in 0.05 (F) and 0.08 (G) M solutions of EMS.

The big tubes turn out to be useless, since they contain cells that were incubated with the wrong DNA after the mutagenesis.  Most of the small tubes also are from stages that have been incubated with DNA, (e.g. label 'F DNA'), but others (the ones labeled '90') were frozen after 90 min of post-mutagenesis growth, before the DNA addition step.  These ones I can use.

The first step now is to do a test I didn't do in the original experiment, to check that the EMS mutagenesis did indeed cause mutations by plate some of the cells on low-concentration novobiocin. I'll do this test on the wildtype cells (B & C), so not to unnecessarily use up the more valuable cells in the marked strains (D-F).  I don't have tubes of the control A culture, so I'll just use normal wildtype cells.

If this test shows that the mutagenesis worked, I have two alternatives.  1.  I could isolate DNA from the mutagenized marked cultures and use it to transform wildtype cells to StrR (D & E, to enrich for cells with sxy mutations) or CmR (F & G, to enrich for cells with murE mutations).  Then I'd enrich these transformants for hypercompetent mutants by transforming them with the PCR'd NovR fragment (after first testing that this works well).  2.  I could do the hypercompetence-selection transformation first, and then isolate DNA and transform wildtype cells with selection for the linked marker.  The advantage of 1 is that I can pool many thousands (millions?) of transformants, maintaining whatever genetic diversity my mutagenesis has created in the gene of interest.

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