So I've completed my big transformation experiment as described in previous posts. Here's the resulting data:
The first two graphs show the difference in transformation frequency between strains and under different conditions. The second pair of graphs shows the ratio between transformation frequency in hfq+ and hfq- strains.
A few things stand out and I would like to pursue them.
1) The sxy-1 hfq- double mutant apparently has a higher transformation frequency than the single sxy-1 mutant. This is striking. I would like to see if this is consistent across other sxy point mutations. I'm in the process of making a sxy-2 (hypercompetent) hfq- mutant to see if this is consistent. I would also like to construct a sxy-7 (decreased competence) hfq- mutant and see the effects of strengthening the mRNA structure because I'm starting to think there may be some regulation happening at the RNA level. The problem with sxy-7 is that it decreases the natural transformability of the cell, so it's much harder to get the hfq knockout into it's genome. I'll see if I can find some way.
2) For some reason, in the simple hfq- strain, there appears to be a consistent TF across all conditions except MIV and log + cAMP. Something must be going on to cause the TF to fall after adding cAMP as the culture gets dense. I would like to do a time-course experiment for the hfq- mutant and perhaps try adding the cAMP at different times to see what happens.
3) I would also like to try and see where the effect of hfq is happening on the competence pathway. Back in the 90's, someone in the lab created a strain that expresses beta galactosidase in the presence of the CRP-cAMP complex. I would like to see how the expression of this reporter gene changes in the hfq knockout verses an hfq+ strain. I am currently growing the hfq+ version of this strain and I plan on transforming hfq- DNA into it.
That'll give me something to do.
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