Measurement

During our project we employed several different measurement techniques to evaluate the performance of our parts:

For in vivo characterization of PANTR1 toehold switches in E. coli, as well as for the InterLab studies, we used a plate reader and simultaneously recorded the OD_600nm and the fluorescence of GFP, mCherry, RFP or BFP.
For the bioproduction of canthaxanthin and of phenazine-1-carboxylate (PCA), we employed high pressure liquid chromatography (HPLC) to evaluate the amount of the compounds produced.

Here we describe the calibrations we performed and the methods we developed for achieving accurate measurements.

Fluorescence Measurements in 96-well plates

Growth experiments of E. coli cells were performed in a CLARIOstar (BMGLabtech) plate reader using an opaque wall 96-well polystyrene microplate, the COSTAR 96 (Corning).
The OD_600nm arbitrary units, recorded to estimate the concentration of bacteria, were converted into standard Particle Count units, while the green, red and blue fluorescence values into Molecules of Equivalent FLuorescein (MEFL), Texas Red (METR) and Cascade Blue (MECB). For this, we performed the calibration of our experimental setup using the iGEM InterLab 2022 calibration protocols.

OD_600nm calibration for a 96-well plate

We were unable to perform the OD_600nm calibration using the NanoCym monodisperse silica nanoparticles provided with the 2022 Distribution's measurement kit. The suspension was crystal clear and the OD_600nm were at the same value as pure water.
To circumvent this obstacle, we used dry Monodisperse Silica Nanoparticles with a diameter 950 nm from Nanocym that we resuspended in water to reach a suspension of 3x10¹⁰ particles / mL (the calculations for silica beads suspension were performed using the online tool developed by the iGEM 2020 Evry Paris-Saclay team). This suspension was further diluted 10 fold and used as a stock of NanoCym 950nm monodisperse silica nanoparticles as indicated in the iGEM InterLab 2022 calibration protocol.
Moreover, we performed measurements before and after step 27 of the protocol, and thus we calibrated our instrument at both 100 µL and 200 µL volume per well.
The results are presented in Figure 1. They indicate that our 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.

Particles standard curve

Figure 1. Particle standard curves used to convert OD_600nm values to the number of particles in suspension.

Green, red and blue fluorescence calibrations for a 96-well plate

We successfully calibrated our experimental setup to detect green, red and blue fluorescent according to the iGEM InterLab 2022 calibration protocol, at both 100 µL and 200 µL volume per well, by performing the measurements before and after steps 26 and 27 of the protocol.
The results presented in Figures 2, 3 and 4, show a linear relationship between measured fluorescence values and the concentration of each compound.

Figure 2. Fluorescein standard curves used to convert arbitrary fluorescence units (λ_excitation 488 nm and λ_emission 530 nm) into Molecules of Equivalent FLuorescein (MEFL).

Figure 3. Texas Red / Sulforhodamine 101 standard curves used to convert arbitrary fluorescence units (λ_excitation 561 nm and λ_emission 610 nm) into Molecules of Equivalent Texas Red (METR).

Figure 4. Cascade Blue standard curves used to convert arbitrary fluorescence units (λ_excitation 405 nm and λ_emission 450 nm) into Molecules of Equivalent Cascade Blue (MECB).

Summary

Using the linear ranges of these standard curves (Figures 1-4), we computed the Particles / Abs600, MEFL / a.u., METR / a.u. and MECB / a.u. ratios and subsequently the coefficients to transform the arbitrary fluorescence / OD_600nm into MEFL / Particles, METR / Particles and MECB / Particles (Table 1).

Table 1. Computed Particles / Abs600, MEFL / a.u., METR / a.u., MECB / a.u. and (MEFL x Abs600) / (a.u. x Particles), (METR x Abs600) / (a.u. x Particles) and (MECB x Abs600) / (a.u. x Particles) values.

	100 µL volume / well	200 µL volume / well
Particles / Abs600	3.32E+08	2.97E+08
MEFL / a.u.	2.61E+09	2.92E+09
METR / a.u.	6.77E+08	6.98E+08
MECB / a.u.	2.97E+09	3.18E+09
(MEFL x Abs600) / (a.u. x Particles)	7.87	9.82
(METR x Abs600) / (a.u. x Particles)	2.04	2.35
(MECB x Abs600) / (a.u. x Particles)	8.92	10.69

Compound quantifications by high pressure liquid chromatography (HPLC)

High pressure liquid chromatography (HPLC) is a technique used to separate compounds based on their different properties. Our host lab is equipped with a Shimadzu Prominence LC20/SIL-20AC HPLC instrument linked to an UV–Vis detector. A Kinetex XB-C18 reversed phase column (250 mm × 4.5 mm, 5 μm) was also available.
As our compounds of interest were hydrophobic, reversed phase chromatography was the method of choice for separating them. Moreover, they are all capable of absorbing light, so spectroscopy was adapted for detecting them.

HPLC method development for PCA detection and quantification

Various methods for reversed phase HPLC separation of phenazine-1-carboxylate (PCA) were described in the literature, but the method of Liu et al. [1] seemed the closest to our host lab routine which uses the Kinetex XB-C18 reversed phase column (250 mm × 4.5 mm, 5 μm) with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. Thus, the flow rate was set to 0.4 mL/min and a Liu et al. -like gradient method was implemented: 0–5 min: 5% B to 50% B; 5–13 min: 50% B to 100% B; 13–23 min: 100% B, 23-25 min: 100% B to 5% B; 60 min, stop. PCA was readily detected at 250 nm with a retention time of 16.4 minutes (Figure 5A).
However, a good HPLC separation method should be able to distinguish between closely related compounds. For this reason, we analysed along with PCA an equal amount of phenazine (Figure 5B). These two compounds were neatly separated: two distinct peaks appeared on the chromatogram (Figure 5C), but only 0.6 minutes apart. In order to increase the distance between these two peaks, we tested different other slopes of the gradient. We observed that a slower hydrophobicity increase leads to a better separation (Figure 5D to 5H), but an upper limit of 1.5 minutes seems to be reached. To further increase this difference, we set-up a two step hydrophobicity increase as in the method described by Liu et al., with a fast (5 minutes) increase to 50% B followed by a much slower increase to 100% B. Thus, the difference in the retention times of PCA and phenazine increased to 2 minutes (Figure 5I).

Figure 5. HPLC method development for an efficient separation of PCA and phenazine. The hydrophobic B phase % concentration gradient is presented in cien blue. Chromatogram images were produced using the Shimadzu’s LabSolutions Postrun Analysis software.

Using this final HPLC separation method (0–5 min: 5% B to 50% B; 5–35 min: 50% B to 100% B; 35–45 min: 100% B, 45-50 min: 100%B to 5% B; 60 min) and increasing concentrations of PCA and phenazine, we established a standard curve for PCA quantification (Figure 6). Up to 100 µM, a linear relationship between the integrated peak areas and the PCA concentration was observed, but at higher concentrations, saturation appeared (hyperbolic curve).

Figure 6. Standard curve for PCA quantification. Full chromatogram representation of PCA and phenazine at increasing concentrations (A) and their zoom on the PCA and phenazine peaks (B). The integrated peak areas are represented as a function of PCA concentration in normal (C) and log scales (D). Images and peak integration were produced using Shimadzu’s LabSolutions Postrun Analysis software.

HPLC method development for carotenoids detection and quantification

In our project we produced different carotenoids (canthaxanthin, ß-carotene, lycopene) that are hydrophobic compounds. So, they are also suited for reverse phase chromatography separation.
Using our host lab routine experimental set-up consisting of the Kinetex XB-C18 reversed phase column (250 mm × 4.5 mm, 5 μm) with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B, we were unable to scale down to less than 60 minutes the elution time.
Nevertheless, by changing the mobile phases A and B to methanol and acetonitrile respectively, we adapted the method described by Afonso et al. [2] and efficiently separated the 3 carotenoids isocratically at a flow rate of 1 mL/min and with an A:B ratio of 90:10.
Standard curve of canthaxanthin (figures 7) shows a linear relationship between the integrated peak areas and the compound concentration within the working ranges of our E. coli extracts. For ß-carotene (figure 8) and lycopene (figure 9) this linearity is observed up to 125µM and 30 µM respectively, whereas at higher concentrations, saturation appears (hyperbolic curve).

Figure 7. Standard curve for canthaxanthin quantification. Full chromatogram representation of canthaxanthin at increasing concentrations (A) and its zoom on the canthaxanthin peaks (B). The integrated peak areas are represented as a function of canthaxanthin concentration in normal (C) and log scales (D). Images and peak integration were produced using Shimadzu’s LabSolutions Postrun Analysis software.

Figure 8. Standard curve for ß-carotene quantification. Full chromatogram representation of ß-carotene at increasing concentrations (A) and its zoom on the ß-carotene peaks (B). The integrated peak areas are represented as a function of ß-carotene concentration in normal (C) and log scales (D). Images and peak integration were produced using Shimadzu’s LabSolutions Postrun Analysis software.

Figure 9. Standard curve for lycopene quantification. Full chromatogram representation of lycopene at increasing concentrations (A) and its zoom on the lycopene peaks (B). The integrated peak areas are represented as a function of lycopene concentration in normal (C) and log scales (D). Images and peak integration were produced using Shimadzu’s LabSolutions Postrun Analysis software.

Conclusions

We were successful in calibrating our experimental setups for the in vivo measurements in 96-well plates not only for the commonly used green fluorescence detection, but also for red and blue fluorescence.
As measurement does not always mean using fluorescence, we developed quantitative methods for measurement of several compounds using reverse phase high pressure liquid chromatography.

References

[1] Liu Y, Zhou Y, Qiao J, Yu W, Pan X, Zhang T, Liu Y, Lu S-E. Phenazine-1-carboxylic acid produced by Pseudomonas chlororaphis YL-1 is effective against Acidovorax citrulli. Microorganisms (2021) 9: 2012.
[2] Afonso BS, Azevedo AG, Gonçalves C, Amado IR, Ferreira EC, Pastrana LM, Cerqueira MA. Bio-based nanoparticles as a carrier of β-carotene: production, characterisation and in vitro gastrointestinal digestion. Molecules (Basel, Switzerland) (2020) 25: E4497.