GFP fluorescent protein is one of the most widely used report genes in synthetic biology. We used it in our project to evaluate the performance of the toehold switches designed to be specific for rosewood, but also for the [“Contribution”](https://2020.igem.org/Team:Evry_Paris-Saclay/Contribution) and [“Improvement”](https://2020.igem.org/Team:Evry_Paris-Saclay/Improvement) parts.
In fact, we used the superfolder GFP (sfGFP) which is a derivative of the Green Fluorescent Protein from *Aequorea victoria* with improved features in terms of intrinsic brightness, tolerance of circular permutation, resistance to chemical denaturants and folding kinetics at 37°C [](#ref1). The protein contains 13 mutations compared to the wild-type (Uniprot P42212): S2R, S30R, Y39N, F64L, S65T, S72A, F99S, N105T, Y145F, M153T, V163A, I171V & A206V. In addition, sfGFP was fused at the C-terminus with the LVA degradation tag (AANDENYALVA), an ssrA tag that accelerates protein degradation in *Escherichia coli* at 37°C [](#ref2) and that is used in synthetic biology to rapidly degrade reporter genes and therefore better observe the synthetic gene networks’ dynamics.
We performed the experiments in two types of conditions: *in vivo*, using *E. coli* cells and *in vitro*, in a cell-free system derived from *E. coli*.
## GFP calibration for a 96-well plate
The *in vivo* experiments using *E. coli* cells were performed in a CLARIOstar (BMGLabtech) plate reader using an opaque wall 96-well polystyrene microplate, the COSTAR 96 (Corning).
To be able to convert the OD600nm and the arbitrary GFP fluorescence units into Molecules of Equivalent FLuorescein (MEFL) / particle, we performed the calibration of our experimental setup using the iGEM calibration protocols:
- [iGEM 2020 Plate Reader Fluorescence Calibration](https://www.protocols.io/view/calibration-protocol-plate-reader-fluorescence-cal-6zrhf56)
- [iGEM 2020 Plate Reader Abs600 (OD) Calibration](https://www.protocols.io/view/calibration-protocol-plate-reader-abs600-od-calibr-bht7j6rn)
This year, due to the COVID-19 pandemic, the iGEM Measurement kit was not provided and a key component was thus missing: the silica beads suspension required to convert the OD600nm into the number of particles.
As per iGEM measurement Committee’s suggestion, we purchased the Monodisperse Silica Nanoparticles with a diameter 950 nm from Nanocym. To prepare the 3x1010 particles / mL solution, usually distributed in the measurement kit, we performed calculations and designed the tool described hereunder. We also provide to the community a modified template [excel](https://2020.igem.org/wiki/images/1/10/T--Evry_Paris-Saclay--iGEM_Data_Analysis_Template-Fluorescence_Standard_Curve_96-well_plate_template.xlsx) file containing all details of those calculations.
### Silica Beads Calculator
to prepare the 3x1010 particles/mL suspension, usually distributed in the iGEM measurement kit
Spheres diameter (nm)
SiO2 density (g/cm3)
(Number supplied by manufacturer)
One Sphere volume (mL)
One Sphere weight (g)
Weight a few tens of mg of silica beads
Volume of ddH2O to be addes (mL)
The row data of our calibrations are provided as an [excel](https://2020.igem.org/wiki/images/4/4c/T--Evry_Paris-Saclay--iGEM_Data_Analysis_Template-Fluorescence_Standard_Curve_96-well_plate_Evry_Paris-Saclay.xlsx) file and the results presented in Figures 1 and 2.
The measurements were subjected to a self-validation using the [Matlab tool available on GitHub.](https://github.com/iGEM-Measurement-Tools/Excel_Process_Validator)
The fluorescence values passed the validation:
- Sufficient dynamic range of fluorescein calibration value: 3569.52
- Found a sufficiently long fluorescein dilution slope from column 1 to 11
- Computed mean MEFL / a.u. is positive
However, the validation failed for Abs600:
- Dynamic range of Abs600 calibration values 17.39 is not at least 20-fold
- Found a sufficiently long fluorescein dilution slope from column 1 to 6
- Computed mean particles / Abs600 is positive
- Found 24 wells with apparently low cell counts
The CLARIOstar (BMGLabtech) plate reader does not give much sensitivity to low values of Abs600 under about 0.1, but we can reliably measure above 0.1.
**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://2020.igem.org/wiki/images/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://2020.igem.org/wiki/images/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.
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.
 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.
 Purcell O, Grierson CS, Bernardo M di, Savery NJ. Temperature dependence of ssrA-tag mediated protein degradation. Journal of Biological Engineering (2012) 6: 10.