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.  

Description

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