Field of Science

R. capsulatus growth curves in RCV medium

My upstairs GTA colleague and I were surprised that the Bioscreen growth curves in the previous post didn't show a dip in OD600 of the GTA-overproducer strain like that seen in manual (non-automated) growth curves.  This dip is thought to be caused by the lysis of GTA-producing cells as GTA production peaks when cells hit stationary phase.

We thought part of the problem might be that I used the standard YPS medium which is based on modest concentrations of yeast extract and peptone.  The clearest/most-recent published demonstration that GTA-producing cultures used RCV, a simpler 'defined' medium based on malate, and showed that the apparent lysis occurred in medium with 0.5 mM PO4 but not in medium with 10 mM PO4. 


So I redid the growth curves for all 6 strains, using both high-P and low-P versions of RCV (kindly supplied by my upstairs colleague).  The results are not inconsistent with the Westbye results, but they're not at all compelling.  None of the strains decreases in OD600

The problem is that there's quite a bit of between-strain variation in growth and in the stability of the stationary phase OD.  (Within each strain the replicate wells give very similar results, with one exception.)

The graph below shows growth in the high-phosphate medium.  The main graph shows OD600 on a log scale (appropriate to exponential growth), and all the strains appear to stably reach similar densities.  But the inset shows the same data on a linear scale, which makes the variation look more significant.  The overproducer strain stops growing abruptly at OD600 = 0.7 a lower density than the other strains.


Here's the cells in the low-phosphate medium.  There's an initial drop in OD600, over the first 10 hours, but then all the strains grow steadily except strain YW1, where the individual wells grew at different rates for no apparent reason.  Again the linear-scale inset shows the substantial variation at stationary phase.  The overproducer DE442 again stops growing, this time at OD600 = 0.8, and now its OD falls by about 20% over the next 40 hours.


I really don't feel comfortable drawing any solid conclusions from this one experiment, especially since there's a blip in many of the growth curves at a point where I stopped and restarted the runs to add more time when I realized that 3 days wasn't going to be long enough.  Even though the shaking only stopped for 2-3 minutes, and the trays of cells remained in their holder with the lid closed, most of the strains had an abrupt change in OD600.  (You can see the blips at hour 63.)

Plan:  Do the run again.  This time I'll pre-grow the cells into log phase in high-P and low-P RCV.  medium (the upstairs colleague has offered me enough medium to do this).  And I'll plan on pausing the run at key times to take samples that I can assay for GTA production.



No comments:

Post a Comment

Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="http://www.fieldofscience.com/">FoS</a> = FoS