Revision as of 15:16, 17 October 2018

Alternative Roots

InterLab Study

Calibration

Alternative Roots participated in the 2018 Interlab study. Three calibration steps were carried out prior to any experimental measurements being taken. Absorbance and fluorescence values were measured in 96-well plates using a Thermofisher Varioskan Lux plate reader (Thermofisher Scientific). Absorbance was measured at 600 nm and converted to a comparable OD₆₀₀. Fluorescence was measured at 525 nm with excitation at 485 nm with a 12 nm bandpass width. All readings took place at 25 °C and pathlength correction was disabled. The calibrations were:

1. A LUDOX CL-X 45 % colloidal silica suspension was used to calculate a conversion factor for the Abs₆₀₀ value measured by the plate reader to a comparable OD₆₀₀ value, considering path length and well volume (Table 1). Abs₆₀₀ of 1:2 dilutions of LUDOX silica suspension were taken in triplicate and a reference OD₆₀₀ of 0.063 (the reference value for 100 µL of LUDOX CL-X in a well of a standard 96-well flat-bottom black with clear bottom plate) divided by the mean measured value to give a conversion factor.

Table 1. Optical density readings for LUDOX CL-X 45% colloidal silica suspension and water used to calculate the conversion factor for absorbance readings to OD₆₀₀ readings for plate reader measurements.

2. A standard curve was prepared by measuring the OD₆₀₀ of serial dilutions of monodisperse silica microspheres, with similar light scattering properties to E. coli cells. This was used to standardise OD readings across labs (Figure 1A).

3. A fluorescence standard curve was created by measuring the fluorescence of serial dilutions of the small molecule fluorescein. This has similar excitation and emission characteristics to GFP allowing conversion of fluorescence readings to an equivalent fluorescein concentration. Calibrations allowed expression measurement in units of fluorescence per OD and molecules of equivalent fluorescein (MEFL) per cell (Figure 1B).

Figure 1. Curves used to calibrate A; Fluorescein per OD using dilutions of fluorescein and B; molecules of equivalent fluorescein per particle using dilutions of solutions of monodisperse silica microspheres.

Protocol

Following these calibration steps, two transformed colonies for each test device and both control plasmids were used to inoculate lysogeny broth (LB) medium containing 25 µg/L of chloramphenicol (CAM) and incubated overnight at 37 °C with shaking at 220 rpm. Overnight cultures were diluted 1:10 and the OD₆₀₀ adjusted to 0.02 with LB with CAM to a final volume of 12 mL. Fluorescence and Abs₆₀₀ were taken at 0h and 6 hours of incubation at 37 °C with 220 rpm shaking. Test devices, plasmid backbone and protocol workflow are shown in Figure 2.

Figure 2. A) test devices transform into DH5α for the iGEM interlab study. A negative control device consisting of a tetracycline resistance gene (BBa_R0040) with no associated promoter, RBS or terminator sequences was also transformed into DH5α. B) All test devices were transformed into DH5α using the pSB1C3 plasmid backbone with chloramphenicol for selection. C) Interlab study protocol workflow.

Results

Results for OD₆₀₀ and fluorescence measurements after 6 hours are shown in Figure 3.

Figure 3. OD₆₀₀ (A) and fluorescence (B) interlab study results for positive and negative controls and all test devices after 6 hours. Tests were performed in duplicate using two colonies for each test device, results for which are shown separately to account for colony-to-colony variation.

Results for both 0 and 6 hours were converted into fluorescence per OD₆₀₀ and fluorescence per cell using the previously described calibrations (Figure 4). While the InterLab study is an opportunity for crowdsourcing a large amount of data on the inherent variability of a given biological system, it does not account for variations within data sets. Our team attempted to seek out and address such sources of variation, investigating biodesign automation of protocols, use of internal standards and effects of media composition. Further detail and results are available on the measurements page.

Figure 4. iGEM interlab study results for positive and negative controls and all test devices. Fluorescence was measured at 525 nm and readings were taken at 0 hours and 6 hours in a 96 well plate. A: Fluorescence per OD₆₀₀, B: Fluorescence per Escherichia coli cell, calibrated to a standard curve of LUDOX silica beads.