Field of Science

Disappointing result

The detailed time course of beta-galactosidase activity in the E. coli strain with the ppdA::lacZ fusion confirmed the results I posted a few days ago. But I'm not going to post these results, because they are superseded (made uninteresting?) by the results of the next experiment.

Having shown that beta-gal activity decreases as cells enter exponential growth, and increases once growth stops, I needed to find out whether these changes depended on the presence of the transcription activators Sxy and CRP. If the answer is yes, then they are due to changes in the activity of the ppdA gene's CRP-S promoter. If the answer is no, then the beta-gal changes are due to something much less interesting (from my present perspective of wanting to find out how E. coli CRP-S promoters are activated). Unfortunately the answer seems to be NO.

Here's the data. The top graph shows culture density as a function of time for the three strains I tested. The first points (t=0) are before the cells were diluted 300-fold. You can see the 'lag' for the first ~30 minutes, then the cells begin growing exponentially. (You can tell that they're doubling at a constant rate because the points fall on a straight line on this log scale.) After about 200 minutes growth slows. The lines aren't joined to the last points because there's a 700 minute gap separating them (this part of the graph isn't to scale). You can also see that one strain grows slower than the others (red line and points); this is the strain whose crp gene is knocked out.

The second graph shows the amount of beta-gal activity in the cultures. Some of the values for the crp- strain (again the red line and points) are a bit low, perhaps because of its slow growth. However the sxy+ and sxy- strains show almost identical patterns (black and blue lines respectively). This means that the changes in beta-gal activity do not depend on Sxy, and thus almost certainly do not reflect changing activity of the ppdA gene's CRP-S promoter. Again the last points are for samples taken after 1200 minutes, when the cultures had been in stationary phase for quite a while.

What could cause the changes in beta-gal activity? One possibility is that the number of copies of the ppdA::lacZ plasmid per cell might continue to increase after cell growth slows. Another is that the lacZ mRNA might be more stable in stationary phase than other mRNAs. There are probably other possibilities too. The only important possibility is that we're wrong about Sxy and CRP being transcriptional activators of this promoter.

What next? I'm right now testing whether the stronger induction seen when the crp+ sxy+ cells were transferred to starvation conditions depends on CRP and Sxy. I hope it does.

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