Team:Evry Paris-Saclay/Measurement
**Figure 1.** Particle standard curve used to convert OD600nm values to the number of particles in suspension.
**Figure 2.** Fluorescein standard curve used to convert arbitrary fluorescence units (λexcitation 483 nm and λemission 530 nm) into Molecules of Equivalent FLuorescein (MEFL).
## GFP calibration for a 384-well plate
The *in vitro* cell-free experiments were performed in a Synergy HTX (BioTek) plate reader using an opaque wall 384-well polystyrene microplate (Nunc 242764).
Only the GFP fluorescence was recorded and, to be able to convert the arbitrary units into Molecules of Equivalent FLuorescein (MEFL), we adapted the 96-well iGEM calibration protocol ([iGEM 2020 Plate Reader Fluorescence Calibration](https://www.protocols.io/view/calibration-protocol-plate-reader-fluorescence-cal-6zrhf56)) to a 384-well experimental setup.
As the cell-free technology is becoming increasingly popular, we provide to the community the corresponding 384-well modified template [excel](https://static.igem.org/mediawiki/2020/f/f3/T--Evry_Paris-Saclay--iGEM_Data_Analysis_Template-Fluorescence_Standard_Curve_384-well_plate_template.xlsx) template.
The row data of our calibrations are available as an [excel](https://static.igem.org/mediawiki/2020/a/ad/T--Evry_Paris-Saclay--iGEM_Data_Analysis_Template-Fluorescence_Standard_Curve_384-well_plate_Evry_Paris-Saclay.xlsx) file and the results are presented in figure 3. The fluorescein standard curve shows a linear relationship between the arbitrary fluorescence values and the compound concentration up to 0.313 µM. At higher concentration values, we observe saturation. This is due to the fact that the plate reader’s settings were adapted to have a more accurate measurement of fluorescence at low concentration values.
**Figure 3.** Fluorescein standard curve used to convert arbitrary fluorescence units (λexcitation 483 nm and λemission 530 nm) into Molecules of Equivalent FLuorescein (MEFL) in a 384-well experimental setup.
## Conclusions
We were successful in calibrating our experimental setups both for the *in vivo* measurements in classical 96-well plates, and also for the *in vitro* cell-free system measurements in 384-well plates. Thus, we bring to iGEM new standardization methods that are particularly useful in the context of diversification of synthetic biology chassis and the use of cell-free systems.
## References
[1] Pédelacq J-D, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology (2006) 24: 79–88.
[2] Purcell O, Grierson CS, Bernardo M di, Savery NJ. Temperature dependence of ssrA-tag mediated protein degradation. Journal of Biological Engineering (2012) 6: 10.