GFP /RFP measurenments

[ReynelUrreaC86]

Dear community,

First, thank you for the information posted. It has helped before to solve some issues. Now, I want to ask if you have experienced the same issue or have a potential solution for the one below.

In a recent experiment, we grew the same yeast strain expressing GFP reporter construct stably integrated in the genome in 5 bioreactors. We were expecting similar values for all bioreactors. However, in both experiments, one of the bioreactors always displayed much higher values than the others (despite having similar OD values to the others). For us, this makes the data interpretation difficult, since we work with promoter engineering and use GFP/RFP signals to compare promoter strengths (resulting in different expression levels of GFP).

I would like to know if these strong differences in signal detection are common between the bioreactors (despite using the same strain, setup). In addition, we wonder if we can fix the problem (e.g. any type of calibration?).

I have contacted the company assembling the bioreactors and they suggested the following: “Our technical team has pointed out the below points:
– Source of error: Alignment of Laser/spectrometer:
Recommendation: Carefully bend Laser/spectrometer until the value gets better. Proceed to use a nonconductive material for bending, else disassemble the sidewalls of the
reactor (not recommended). >I did this, but it does not help.

Many thanks for any help / comment about this issue.

Sincerely,

Reynel

[harrison]

Hello,
THe answer is there ARE differences between reactors and how important they are depends on the biological system + media + fluorescence signal + choice of excitation/emission bands. FOr example, each of the LEDs and each of the spectrometers have slight differences in manufacturing, and the “zero” level of the signal can depend very sensitively on these, so it is important to calibrate it out.

GENERALLY in our lab what we do is measure a “zero” signal for each reactor. For example putting the same non-fluorescent sample in each (i.e. just cells +media) , do the measurement, record the value as the “zero” value. Then, we subtract that zero from the experimental data to bring them to a common baseline. Doing this we find we can get very reproducible results, though admittedly it is much easier if you have a strongly expressed fluorescent protein and limited background (e.g. a non-fluorescent media)
Best wishes
Harrison

[ReynelUrreaC86]

Dear Harrison,

thank you for the clarification and idea. I will check this.

In addition, I take the opportunity to ask an additional question. I am not a programmer, but have a bit of experience on Python. With the Chi.Bio, I want to implement pulses (10 s every 5 min for 2 hours) with the red-light LED to induce expression. I know I can implement this as custom program, but after checking the Phyton script, I am not sure how to add/modify the algorithm. Ideally, I would activate the LED between the measurements. I will be working on this, but maybe there is a quick help/advise from your side.

Best wishes,
Reynel

[harrison]

Hello
There is already an optogenetics mode which is basically doing this. It is not set up to run 10s every 5 minutes, but rather at variable intensities during each 1 minute cycle. Do you explicitly need the 5 minute cycle or are other ways OK?

In general if you want to implement from scratch, then yes, custom program is fine. I would look at the optogenetics one to use as a template for how to switch LEDs as and when needed.

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