Team:Newcastle/InterLab
Alternative Roots
InterLab Study
2018 InterLab Study Aims
A weakness in the measurement of fluorescence relative to optical density (OD), as with previous IGEM interlab protocols, is the potential discrepancy between optical density and actual cell concentration. This year the iGEM study aims to reduce lab-to-lab variability further by measuring GFP fluorescence relative to absolute cell counts or colony forming units. Normalisation of fluorescence to colony forming units goes further by allowing measurement of fluorescence relative only to viable cells, and thus a more accurate measurement of promoter strength, whereas OD600 and absolute cell count measures cannot differentiate between viable and non-viable cells.
Calibration Steps
Three calibration steps were carried out prior to experimental measurements being taken:
First, 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. 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.
Secondly, 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, used to estimate cell numbers (Figure 1 A).
Thirdly, a fluorescence standard curve was created by measuring the fluorescence of serial dilutions of the small molecule fluorescein, which has similar excitation and emission characteristics to GFP. This allowed 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).
InterLab Protocol
Following calibrations, two transformed colonies for each test device and both controls were used to inoculate LB medium containing 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.
Abs600 analysis of each test device
All test devices produced growth to OD600 reading in excess of 0.3, except test device (TD) 4. Despite lower growth than other transformants, TD4 produced the highest mean fluorescence reading of 79.1 a.u., as was expected as the strongest promoter of the Anderson collection (parts.igem.org/Promoters/Catalog/Anderson). Figure 3A and 3B show the colony 1 and colony 2 Abs600 values for the controls and each test device at 0 hours and 6 hours respectively.
Description
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