Just out in OUP Synthetic Biology, "Multicolor Plate Reader Fluorescence Calibration" extends our prior work on calibrating green fluorescence and cell count to calibrate red and blue fluorescence as well. The results are no surprise (if we can use a green dye, we ought to be able to use other dyes too), but it's valuable to have specific recommendations for dyes to use and to have an interlab study validate that yes, they really do perform as well as the others.
So everybody out there listening, please start using sulforhodamine-101 to calibrate your red fluorescence and Cascade Blue to calibrate your blue fluorescence! Everybody who uses your data will thank you for providing equivalent molecule/cell estimates rather than irreproductible arbitrary or relative units.
|Red and blue fluorescence calibrants were just as precise as the prior green and cell-count calibrants|
The paper also reports on some of the travails we ran into making the study work: some of the fluorescent proteins we wanted to try out didn't work in our hands, and there were miscellaneous other problems: a promoter sequence got messed up, some things wouldn't synthesize, one of the plasmids seemed problematic, and timing problems meant not all labs could run all constructs.
Problems like that are frustrating, but ultimately I'm happier reporting them than burying them. Remember: if you read a synthetic biology study with lab work and it doesn't talk about failures, it just means they either aren't aware of them or else they've pruned them from the narrative! Calibration methods like these help us see better when things go wrong and understand what's happened.