Hello,
Baseband normalisation is essentially calibrating for the excitation intensity as well as OD. Therefore, the baseband-normalised data should stay approximately constant if one was to hold fluorescence fixed and change OD.

If you are allowing BOTH OD and GFP/mScar to change at the same time, then you MIGHT see a reduction in fluroescence essentially because the fluorescence/cell is decreasing. This often happens if you have a system in which it has grown overnight (i.e. to high fluoprescence stationary state) then you set it going back to exponential phase in the morning (i.e. diluted into fresh media), after which the cells which previously had time to achieve massively high fluorescence/cell are now having this diluted out.

I would say in general by far the best results come from keeping OD constant (i.e. in turbidostat mode) then looking at changes in fluorescence.

You could adjust the software to show you non-normalised data if you like. Have you taken a look at the section that discusses normalisation in the Supplementary of the paper?

One other thing – if you use LB you will get generally a TERRIBLE signal (i.e. in terms of signal-noise ratio) due to the fact that it is very auto-fluorescent and generally also has large variability batch-to-batch and within a batch over time. Potentially unless you have VERY fluorescent cells this kind of chaos will completely obscure the actual fluorescence changes youw ant to measure. I would strongly recommend using M9 media at least…

One thing I often observe is a fluorescence DECREASE at the start of experiments if inoculating with cells that have come for stationary phase, or even from some other condition where fluorescence levels might be high. Even for promoters that are theoretically uninduced, often at stationary phase there is a lot of leakage, and if they haven’t been diluted this gives lots of time for them to accumulate fluorescence. Then, when you put them into a reactor (or plate reader) to grow you see fluorescence decrease initially as this previously built up level is diluted. This is less obvious if you are doing high levels of induction since the (desired) production is greater compared to the leak production.
DO you think this might explain what you are observing? Potentially I misinterpreted your description…

Hello,

Yes, by adding that dash I can now see it.
I think, contrasting your previous post, to me it looks like the EXACT same thing is happening in both the low and medium inductions for early times (i.e. until you start regulating OD). They behave basically the same (i.e. initial decrease), but the medium one is at least induced enough so that you can see it rise (whereas the low one is not enough for this) once they reach fixed OD.

Another possible explanation could be other non-linearities in the normalisation. For example that light scattering properties at the two wavelengths change as the OD changes, hence it appears to go down (slightly) over time. In general, the system won’t be great if measuring either very low fluorescence levels per cells, or measuring fluorescence at very low cell densities, since in both cases the bleed-through of the excitation to emission filters is drowning out the signal.

Generally our team does not find this a problem since usually what we do is grow cells to a fixed OD and hold them there for at least 5-10 generations (so that any dependence on previous culturing or state is mitigated), and THEN add inducer chemicals (or light, whatever). Is there a reason why you are not doing it this way? Trying to separate out the behaviour of genetic circuits from the impact of changing cell density is always challenging – hence why we make turbidostats in the first place.