Friday, May 1, 2009

Can I, too, transform Haemophilus?

To kick things off, I’ve performed the lab’s most basic transformation protocol of Haemophilus, using the reference strain (Rd KW20 or RR722) as a recipient of MAP7 DNA, which contains seven antibiotic resistance markers. My plan was to look at the transformation rates of two linked markers and their co-transformation rates. Ideally, I’d also include a third unlinked marker to measure the frequency of competent cells in the preparation, but that will wait until next time. For now I just wanted to get the mechanics down.

Luckily, my years in the Burgess Lab working with budding yeast seems to have prepared me pretty well for this, so I only have to hassle my new lab mate, Sunita, for help minimally.

The protocol is pretty straightforward:
  1. Grow an overnight culture in sBHI* from frozen stocks;
  2. Subculture in the morning (1:100 and 1:50 dilutions) for a couple hours in sBHI media until the OD(600nm) reaches 0.20 (indicative of log-phase growth);
  3. Wash and resuspend in M-IV** media for 100 minutes to make the cells maximally competent;
  4. Transform with 1 ug of MAP7 DNA (plus a No DNA Control) in 1 ml of competent cells (with remaining aliquots frozen in 15% glycerol) for 15 minutes;
  5. Perform a dilution series and plate to 4 kinds of sBHI media: No antibiotic, +kanamycin, +novobiocin, and +both.
The next day, if all went well, transformants should appear on the plates, and the ratio of antibiotic resistant colonies per viable colony should provide the frequency of transformation of the corresponding genetic mutation from MAP7 to Rd.

My first attempt failed to yield a subculture that reached the appropriate optical density by the end of the day. After several hours, I realized that I was blanking the spectrophotometer with the wrong media (unsupplemented BHI), and upon reblanking with freshly made sBHI, the cultures seemed appropriately dense, but still something was wrong: I went through the remainder of the protocol and plated cells onto media, but the total amount of viable cells observed the next day was two orders of magnitude too low and there were no observable transformants. Obviously, I need to blank with the same media as was used for the subculture. Oops!

Why the cells refused to grow remains a mystery, especially since the overnights seems to grow just fine. Alas.

My second attempt went far more smoothly. I realized that my overnight cultures had a somewhat lower than expected density (~0.7, instead of >2.0 as reported by Maughan and Redfield 2009 for Rd), so I gave the subcultures some extra time without worrying (4.5 hrs, instead of 2.5 hrs). They reached appropriate densities (interestingly, both the 1:100 and 1:50 dilutions had roughly the same OD by this time), went into M-IV for 100 minutes, got transformed ±DNA, and were either diluted and plated or frozen as 1.25 ml aliquots in 15% glycerol. I also did some spot tests on extra plates just for fun.

The next day, I found that I’d plated at far too high of a density to count viable cells, but... TRANSFORMANTS! Perhaps not a flawlessly executed experiment, but at least the phenomenon I came here to study does indeed happen in my own hands. A happy day.

But I drastically overshot the mark on the plain sBHI plates. Furthermore, I’d had some issues evenly spreading new hemin onto the plates (I think they were too dry), so growth was somewhat uneven. As far as the high density goes, possibly my serial dilution was messed up (not changing tips?... I doubt it, unless the cells are very sticky) or the density of cells was initially higher than I thought. So I returned to the frozen competent cell preparations and re-plated to fresh plates, hopefully in range of being able to accurately count transformation and co-transformation frequencies. I’ll have to wait until this afternoon to get a clearer idea of how my plating went. Stay tuned!

* supplemented Brain-Heart-Infusion broth
** starvation media (at least of nucleotides and their precursors)

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