On Saturday I did a time course examining whether the expression of the ppdA::lacZ fusion (and I hope thus of the sxy gene) changed as growth conditions changed while the cells were growing in rich medium (LB) and after transfer to a starvation medium.
The green line shows how the cells grew. First the density fell sharply because I diluted the cells 1/250 in fresh LB (+Amp to maintain the plasmid carrying the fusion). After a brief period of slow growth while the cells adjusted their metabolism to the improved conditions ("lag" phase), the cells grew exponentially, doubling about every 30 minutes ("log" phase). After about 220 minutes growth slowed down as the medium became depleted of nutrients (still growing so not yet in "stationary" phase).
The blue line in the second graph shows the expression of the lacZ fusion in LB. The first sample was from the dense culture (it had been left on my bench overnight). Once those cells were diluted into fresh LB the amount of beta-gal activity slowly fell, presumably because expression of the lacZ fusion decreased. Part of the reason the decrease is slow is that the cells still contain beta-galactosidase that will either be degraded or diluted out by cell growth. The amount of beta-gal activity remained low while the cells grew, until the cell density got quite high (OD600 about 1.0), when the activity began to increase. I know that the low activity reflects lacZ expression by cells in exponential growth, and not residual enzyme from earlier induction, because I diluted part of the log-phase culture 100-fold into fresh LB+Amp, and found the same activity after these cells had spent an additional 2 and 3 hours in exponential growth (blue circles).
But once the cells got so dense that growth slowed, the beta-gal activity increased, and by 300 minutes had become at least as high as that of the overnight cells from my bench. This is a weaker version of the gene induction we see as Haemophilus influenzae approaches stationary phase.
In H. influenzae, rapid transfer to a medium lacking most nutrients causes strong induction of CRP-S genes, including the homolog of ppdA. To mimic this I transfered the log-phase cells to a medium consisting of minimal salts (M9) plus the same small amount of amino acids we use for H. influenzae. The two red bars show that this transfer caused a more dramatic increase in beta-gal activity.
So, these are weak but nice results. They suggest that CRP-S genes in E. coli are regulated by similar factors to those regulating CRP-S genes in H. influenzae.
[Just to check, I also induced lacZ expression in wildtype (lac+) cells by adding IPTG. Even though these cells have only one copy of the lacZ gene (in the chromosome, rather than one on each of many copies of a plasmid), after 60 minutes they expressed 7 times as much beta-gal activity as the starved ppdA cells did after 120 minutes. This is slightly less than the ppdA cells expressed after Sxy was overexpressed from the pASKA plasmid, and suggests that the CRP-S promoter is being only partially induced by my starvation conditions.]
What next? I need to repeat this time course, starting with a proper overnight culture and taking more time points, especially after log phase. I need to get a longer log-phase series too. And a time course in the starvation medium.
What mutation rate do I want for my experiment?
18 hours ago in RRResearch