Synthetic biology requires reliable and predictable models that enable reproducibility of engineered genetic constructs in variable conditions. These models are essential to facilitate the applications of engineered organism in a wide range of areas from medicine and pharmaceutics to environment and agriculture (Beal et al, 2016). The Interlaboratory Study of Reproducibility (Interlab) seeks to integrate results from different labs around the world using standardized protocols. One key step in these protocols is the conversion of relative units into absolute units, to obtain comparable data in order to determinate the variability in results. The objective of this research is the quantification of expression of three genetic constructs under the control of different promoters (J23101, J23106, J23117), using Green Fluorescent Protein (GFP) as a reporter gene in Escherichia coli DH5α.
Competent cells were obtained following the iGEM protocol Help:Protocols/Competent Cells
E. coli DH5α was transformed using the protocol Help:Protocols/Transformation
The Fluorescence Intensity was measured using the standardized protocol from iGEM Plate_Reader_Protocol_Update . The Plate Reader (Fluostar omega, BMG LABTECH) was calibrated using the solutions included in the Interlab Measurement Kit.
Fluorescence Intensity was measured using the Flow Cytometer (Attune NxT, Thermo Fisher Scientific) in cells grown in LB following guidelines from iGEM. The Flow Cytometer was calibrated using Sphero®Rainbow Calibration Particles (BD Bioscience), 8 peaks, calibrated for MEFL (Molecules of Equivalent Fluorescein). Four drops of calibration particles were dissolved in sheath fluid (1ml). Samples were prepared for measurement in the Flow Cytometer washing the culture media in filtered 1X PBS. Cells cultures were diluted 1:100, adding PSB in a 96-well microtiter plate (Thermofisher Scientific). The instrument was configured with a channel for GFP measurement with 488 nm laser and 530/30 filter. Results were analysed in FlowjoTM v10.1r7 and exported to Excel, conversion from relative units to MEFL were calculated using the sheet provided by iGEM.
Figure 1 shows the standard curve for serial dilutions of FITC in the plate reader under the same conditions used to measure fluorescence in cells. The graphs was used to calibrated the equipment and convert from Fluorescence Intensity to molecules of GFP per cell.
Figure 2 indicates how cell population changes over time for 6 hours for each replicate. Absorbance 600 was corrected using the correction factor: 2,0344 and substracting blank values. A significant increase is observed between 2-5 hours for most devices. Device 1 shows a slow cell growth rate over time, however, replicate 1 exhibit a higher growth rate after five hours.
Fluorescence Intensity (fig 3) indicates that device 1 replicate 1 exhibits a notable increase in fluorescence between 5-6 hours. In contrast, Device 2, replicates 1 and 2, shows a steady increase in fluorescence for the six hours.Despite having high growth rates, Device 3 shows the lowest fluorescence Intensity.
Figure 4 shows the expression of GFP per cell as an indirect measurement of level of expression of each construct for six hours, calculated from Fluorescence Intensity and Absorbance. Device 1 has a strong expression compared with device 2 and device 3. However, the precision of fluorescence Intensity measurement diminishes as the level of expression of GFP per cell increases with the time in strong and medium promoters, indicated by the standard deviation in devices 1 and 2 after 6 hours.Sources of variation in cell growth and gene expression may include autofluorescence, nutrient depletion and growth inhibition by toxic compounds (Lichten et al, 2014). Device 3 shows a weak and constant expression throughout the experiment, with low variation in the fluorescence intensity per cell.
Histograms (Figure 5) show the fluorescence intensity in GFP expressed by cells after incubation for 6 hours under the same conditions (37°C, shaking 220 rpm). Cells population were gated to exclude dying or dead cells according to the Forward Scatter-Side scatter diagram. Positive control, Device 1 and Device 2 produce two peaks of fluorescence intensity. The highest intensity is observed in device 1, however, the largest population expressing fluorescence is observed for devices 2 and positive control.Due to the fluorescence intensity, histograms confirm that J23101 (Device 1) is a strong promoter,
Results from the Plate Reader and Flow cytometer are consistent. Device 2 exhibits a larger number of cells but lower Fluorescence Intensity after 6 hour, in constrast, Device 1, which shows slow growth, has the highest Fluorescence Intensity. The lowest Intensity was detected in Device 3 using the plate reader, no fluorescence was detected in the flow cytometer.
Devices with stronger expression, exhibit a high degree of variation between treatments and replicates.
This study comfirms the promoter strength reported by Anderson Promoter Collection. Promoter J23101 is a strong promoter, J23106 is a medium strength promoter and J23117 is a weak promoter when they are expressed in E. coli
Beal J, Haddock-Angelli T, Gershater M, de Mora K, Lizarazo M, Hollenhorst J, et al. (2016) Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. PLoS ONE 11(3): e0150182.
Anderson, C. Berkley iGEM Team. (2006) Anderson Promoter Collection. Registry of Standard Biological Parts. Available at: http://parts.igem.org/Promoters/Catalog/Anderson
Lichten, C A, White, R, Clark, I B, & Swain, P S. (2014). Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers. BMC Biotechnology, 14: 11
