Field of Science

Timing of competence development in a new murE749 transformant

In the previous post I realized that I don't know how quickly competence will develop in cells that have just acquired a murE hypercompetence mutation.  In that post this was a practical problem (how long should the mutagenized cells be incubated for), but it also has research-grade implications.  That's because we still have no idea how these mutations turn on the competence regulon.  If they take a long time to have an effect, I would suspect some sort of gradually developing imbalance that eventually changes Sxy expression, whereas if they act quickly I'd suspect a more direct effect.

How could I find this out?  I would need to introduce the murE749 hypercompetence mutation into a murE+ cell, presumably by transformation, and then follow the development of competence.  I can do this because we have strains with a CmR cassette that's about 70% linked to murE in transformations. Strain RR797 has the cassette and the murE749 mutation; strain RR805 has the cassette and murE+. So if I transform competent wildtype cells with RR797 DNA and select for chloramphenicol resistance, about 70% of the CmR cells should have the murE749 mutation.

If I do this CmR selection in broth (plating on the side just to check that the transformation worked), I can check how long it takes for the culture to increase its log-phase competence from a transformation frequency of <10 10="" 70="" a="" about="" expected="" i="" mixture="" of="" s="" that="" the="" to="">murE749
.  In parallel, as a control that will also provide useful information, I'll transform with RR805 DNA and measure how long the CmR cells and unselected cells take to completely lose competence and resume exponential growth in sBHI at low density.  (I think I've measured loss of competence before but not carefully.)
How long will this take?  I think it's a one-day experiment after I make the DNAs (or if I find old stocks of the DNAs in my fridge (later - found RR805 DNA but not RR797 DNA so I'll plan on remaking them both).

  1. Pour lots of plain, Cm and Cm+Nov plates
  2. Thaw out frozen competent Rd cells
  3. Incubate with DNAs, add DNase I for 5 min
  4. Filter to wash away the DNA and (more importantly) the DNase I
  5. Resuspend in sBHI (t = 0)
  6. Grow 1 hr, add chloramphenicol (1 µg/ml I think)
  7. Take aliquots to tubes with MAP7 DNA at t = 1 hr, 1.5 hr, 2 hr, 2.5 hr, 3 hr (longer?).  
  8. Dilute and plate on plain, Cm and Cm+Nov plates.
Would we learn anything?  Yes if competence development after transformation is very fast (appears as quickly as chloramphenicol resistance) or very slow, no otherwise.

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