We perform measurements to quantify various parameters, compare different parts and evaluate the quality. Negative and positive controls are vital for interpreting measurements in a given experimental setup. Moreover, presenting the results using standard units of measurement allows others to reproduce the experiments and makes experiments performed in different laboratories easily comparable.
Measurement is a central aspect of any synthetic biology project. When direct measurements of native proteins is not possible or difficult, the use of fluorescent or coloured reporters allows us to obtain an easily detectable signal.
Fluorescent proteins are one of the most widely used reportes in synthetic biology and in the iGEM projects, and among them GFP (and its various mutants) are in the first row.
However, fluorescence units are arbitrary and dependent strongly on the experimental setup: instrument and its parameters, sample containers (e.g. type of microtiter plate).
To allow converting data to “universal” units of measurement, calibration protocols were developed for converting:
OD at λ=600nm (that evaluates the number of cells in suspension) into a number of particles with a well defined size
GFP fluorescence arbitrary units to Molecules of Equivalent FLuorescein (MEFL)
However, GFP is not the only fluorescent protein that can be used as a reporter. For our project we used also RedStar2, a red fluorescent protein that is particularly suitable for yeast . To the best of our knowledge, no calibration protocols were available for the red fluorescent proteins when we started this project. Thus, we developed and present here two calibration protocols for red fluorescent proteins that we successfully used for analysing the expression levels driven by a series of pTef1 promoters in two strains of Yarrowia lipolytica (results available on the dedicated page of this wiki: Promoters & Fluorescent Proteins).
GFP is the Green Fluorescent Protein that was initially discovered in Aequorea victoria and subsequently also in some other organisms.
In our project, we used the hrGFP, the humanized form of Renilla reniformis GFP [2, 3], as a codon-optimized version for the yeast Yarrowia lipolytica was already available in iGEM registry
(BBa_K2117001). We have improved the characterisation of this part and added quantitative data of its expression in Yarrowia lipolytica under the control of different versions of the pTef1 promoter, including BBa_K2117005.
We performed the experiments 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:
The row data are provided as an Excel file and the results presented in figures 1 and 2. The iGEM measurement Committee validated these calibrations. It should be noted however that the CLARIOstar (BMGLabtech) plate reader reader is not giving much sensitivity to low values under about Abs600nm 0.1.
RedStar2 (BBa_K2983060) is a derivative of the Red Fluorescent Protein from Discosoma sp. with improved features in terms of intrinsic brightness and speed of maturation . The protein is tetrameric and contains 15 mutations compared to the wild-type sequence (Uniprot Q9U6Y8) : ΔR2, K5E, N6D, K9T, R17K, H41T, N42Q, V44A, V96I, F124L, A145P, M182K, P186Q, T202I, T217A.
RedStar2 emits bright red light upon excitation and was introduced as a the “brightest and most yeast-optimized version of the red fluorescent protein” . It’s for this reason that we chose it for characterization of the pTef1 promoter activity in the yeast Yarrowia lipolytica and improving an existing part in the iGEM registry (BBa_K2117000).
Unlike GFP, for which calibration protocols are available which allow converting data to “universal” units of measurement (MEFL/particle), to the best of our knowledge, this was not the case for the red fluorescent proteins when we started this project.
In the case of GFP, fluorescence calibration is performed using fluorescein, a compound whose excitation and emission wavelengths are similar to those of GFP. For RedStar2 (λexcitation 558 nm and λemission 583 nm), we uncovered two compounds that have similar excitation and emission properties compared to RedStar2 (Table 1).
|Fluorescent Molecules||CAS Number||λexcitation||λemission||Reference|
|Resorufin||635-78-9 & 34994-50-8 (sodium salt)||571 nm||585 nm|||
|Rhodamine B||81-88-9||555 nm||580 nm|||
To carry out the calibration, we followed the iGEM 2019 Plate Reader Fluorescence Calibration
protocol and replaced the fluorescein by either resorufin or rhodamine B. The results are presented in Figures 3 and 4 and the raw data are provided as excel files for both resorufin or rhodamine B.
We performed the experiments in a SynergyMx (BioTek) plate reader using an opaque wall 96-well polystyrene microplate, the COSTAR 96 (Corning). Changing the plate reader compared to the GFP measurements was due to CLARIOstar (BMGLabtech) plate reader characteristics that is unable to perform excitation and emission at close wavelengths. The iGEM 2019 Plate Reader Abs600 (OD) Calibration was used to calibrate this plate reader too. The raw data are included in the fluorescence calibration excel files (resorufin or rhodamine B) and the results are presented in figures 5.
Both resorufin and rhodamine B standard curves (Figures 3 and 4) show a linear relation between the arbitrary fluorescence values and the compound concentration up to 1.25 µM. At concentration values higher than 2.5 μM, we observe saturation. This is due to the fact that the plate reader’s setting were adapted to detect low concentration values and have a more accurate measurement of fluorescence. Indeed, this is required for detecting RedStar2 expression driven by a single gene copy interested into Y. lipolytica genome. As was the case for CLARIOstar (BMGLabtech), the SynergyMx (BioTek) plate reader did not give much sensitivity either to low values under about Abs600nm 0.1.
One of the main concerns related to the calibration for RedStar2 is related to the fact that both resorufin and rhodamine B absorb also at 600 nm. To clarify any doubts about a potential bias on the absorbance measurement, we measured the absorbance of these compounds at 600 nm. If this bias was significant, this bias could have distorted the fluorescence measurement at 600 nm and therefore the estimate of cell growth. Fortunately, we have established an average bias of 0.010 on the measure of resorufin and 0.002 on the measurement of rhodamine B. Thus, such a bias is negligible for our purposes.
The raw data are included in the fluorescence calibration excel files (resorufin or rhodamine B), raw experimental measurements sheet (the plate layout being the same as in the resorufin / rhodamine B standard curve sheets).
We were successful at calibrating our measurement device by applying the iGEM fluorescence protocol on fluorescein and by adapting it for the calibration using two new compounds resorufin and rhodamine B. Thus we bring to iGEM new standardization methods that are particularly useful in the context of diversification of synthetic biology chassis which need fluorescent proteins other than GFP.
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