No NovR colonies at all?

Yesterday I made 8 preps of competent cells, all to further our phenotyping of our new competence-gene mutants.  Four of them needed to have transformation assays done, three were to be frozen for later DNA-uptake assays by the postdoc*, and one was a knockout of the competence-regulator sxy, to be used as a negative control in the uptake assays.

I didn't include a wild-type positive control strain for my transformation assays, because I've done this lots of times before.  But I did assay the sxy mutant, just to confirm that I had the right strain.  The assay is simple: mix 1 ml of competent cells (~10^9 cells) with 1 µg of NovR chromosomal DNA, incubate for 15 min, add 10 µg DNase I, incubate for 5 min, dilute and plate on plain sBHI agar and on sBHI agar with 2.5 µg novobiocin/ml.

Most of the strains I assayed had been tested before, but some with not-very-consistent results, and I was expecting to see a wide range of transformation frequencies (maximum about 5 x 10^-3 and minimum less than 10^-8 (the detection limit)).  Because I wasn't sure what I would find, I made a point of plating 100 µl of undiluted culture from each assay.  BUT, there were absolutely no NovR colonies on any of the plates.

So I'm pretty sure I screwed something up.  But what?  I used the same DNA stock tube I've used many times before, and I definitely remember putting 3 µl of DNA into each assay tube.  I made fresh sBHI + novobiocin plates using pre-made BHI agar,, and I definitely remember adding the hemin (4 ml), NAD (80 µl) and novobiocin (40 µl) to the melted agar before I poured the plates.  The DNaseI should be fine; I've used this tube before.  And the cells aren't dead, as the plain sBHI plates had the expected numbers of colonies.  Oh how I wish I'd included the positive control!  Luckily I froze one tube of competent culture of each of the strains I transformed, 'just in case', so I can redo the transformations without having to make now competent preps.

To check if I somehow screwed up the agar plates despite my 'definite' memory, I've streaked a test known NovR strain on them, with and without more NAD or hemin.  Before I go home I should set up some overnight cultures of the strains I'm going to test tomorrow...  Wait, will I have time to do this tomorrow?  It takes time and planning to get the cells into the right growth stage for the competence treatment, and then a couple of hours for competence development and the transformation assays.  I have a meeting in the middle of the day, and then we're going to finalize the grades for the big genetics course...  Yes, sure, I can always work late.

* The postdoc is getting interesting results from these assays.  First, all of the mutants he's tested, even those lacking genes thought to be essential for DNA uptake bind/take up at least fourfold more DNA than the noncompetent log-phase cells he's using as a negative control.  The competent sxy- cells I've just made won't induce any competence genes in this medium, so the amount of DNA they associate with will tell us whether this binding is just a property of cells that have been incubated in the starvation medium, or represents induction of some competence genes.

For a couple of the mutants, he's found much higher DNA association levels than we expected.  One mutant should lack the secretin pore through which the pseudopilus contacts the DNA; the other lacks PilF2, an 'accessory' pilin that's absolutely required for transformation, presumably because it's absolutely required for DNA uptake.  The next step is to repeat the DNA uptake assays, this time comparing treatments with and without DNase I in the wash step.  If the mutant cells are binding to DNA but not taking it up, the DNase I should remove the DNA.

UPDATE:  My novobiocin plates had no NovR colonies because I had forgotten to add the required hemin supplement to the agar!  How embarrassing - I haven't made that mistake in years.