Field of Science

Transformations with dirty DNA

The previous post considers ways to test the effects on transformation of chromosomal proteins bound to donor DNA, by gently lysing donor cells and transforming recipient cells with the crude lysate.  We've now done one experiment trying out some methods.

We used donor cells resistant to novobiocin.  We tried freezing the cells without adding the usual 16% glycerol as cryoprotectant, vortexing them with a drop of chloroform or in 0.0001% SDS to disrupt the membranes, with or without pretreatment with EDTA and lysozyme (0.1 mg/ml, 1/10 the normal concentration) to break down the cell walls.

One question was how efficiently different methods would kill all the donor cells.  If some donor cells remain alive they will form colonies on the selective plates used to isolate transformants, confounding measurements of transformation.  Freezing was surprisingly effective.  Many aliquots of a single 'late-log' culture (OD600 = 1.0) were originally frozen at -80 °C in their normal growth medium.  After thawing, 20-40% of the cells were still viable.  All the treatments (listed below) decreased viability, but most left 10,000 or more viable cells per ml.  But after the various treatments we froze the cells again, this time at -20 °C, and when these were thawed there were no viable cells.  This is good, because freezing/thawing is very easy and we don't expect it to disrupt the associations of chromosomal proteins with DNA.

We had 5 treatments:

  1. just chloroform
  2. just SDS
  3. lysozyme then chloroform
  4. lysozyme then SDS
  5. just lysozyme

After the various treatments we pelletted the remaining cells and/or debris, and used 10 µl and 100 µl of each sample as 'donor DNA' in transformations of sensitive wildtype cells.  We got LOTS of transformants from all the treated samples, sometimes as many or more than from an equivalent amount of purified DNA.  There were differences between samples but the causes are unclear.

Next steps:

Freezing/thawing:  try just -20 °C with no other treatment.  Does this kill all the cells?  Does it liberate transforming DNA?

Try less SDS plus lysozyme, and try a milder detergent.

To characterize the DNA, try running the treated samples in an agarose gel, say 0.5% agarose so that large DNA enters the gel.  Run samples ± lots of SDS, to see what difference bound proteins might be making.

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