Chi.Bio not detecting fluorescence from GFP,RFP cultures

[harrison]

Hi John,

A few points/questions
– How “fluorescent” are the “appropriately fluorescent” cells? Do you mean they are at some level that (for example) is visible to the eye? How much expression did you measure from them as a fraction of e.g. “the brightest GFP E.Coli you have ever seen”
– You shouldn’t need to manually switching on the excitation LEDs first. When you click “Measure FPs” it will turn them on itself (and then off again ater the measurement).
– For the GFP have you tried exciting with the lower (395nm) LED?

Generally the system works best when you are not taking tubes in and out of the reactors. There is some wiggle room around the tube and this can impact absolute values of readings. Furthermore, when you start/stop stirring and heat/cool the reactor these will all make some (small) impact on the measurement. Instead, what the system is really designed to do is to track a single test tube (and its contained culture) over time. This is what is done in Figure 5 of the PLoS Biology Paper! So, to best assess your setup I would either a) Test with an inducible construct, which you can maintain at a set OD and then induce and observe fluorescence production or b) Grow your GFP cells to a fixed OD and maintain them there for some time, then pipette into the reactor concentrated RFP cells (say, with the goal of the resultant culture being 50% GFP and 50% RFP) and see how this changes the GFP/RFP balance measured over time.

[jmarken]

Hi Harrison,

Thanks so much for your prompt and thorough answer. We’ll try your suggestions and get back to you with the results.

John

[fbertaux]

Hi John and Harrison,

I can comment on our experience with yeast using light-induced expression mNeonGreen (single integrated copy of the promoter, full induction around pTDH3 strong promoter). We can clearly see the fluorescence reading change when switching ON or OFF the induction for a single culture in turbidostat mode. But the signal is low, so raw numbers cannot be compared between reactors. Also the OD matters a lot, even a small ‘dither’ will be visible on the reading. To be able to compare data between different runs and different reactors, we are now testing if a single calibration (specific to a given reactor, OD and media obviously) provides week-to-week reproducibility.

François

[harrison]

Thanks for the update François, that sounds about what I expect – given it is a single copy. One challenge of week-to-week reproducibility for small signals might be movement of the test-tube when you take it in or out. Your best bet might be to have it “auto calibrate” at the start of experiments (or whenever you expect there is ~zero fluorescence) or something like that.

Do your reactors have (white plastic) diffusers in their spectrometers? There could be a small white sheet just behind the apperture of the spectrometer (i.e. the circular part with hole in the middle). If not, I can mail you some – they can significantly improve signal-to-noise for small measurements (mostly it improves OD measurement, but also fluorescence to some extent).

Harrison

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