| Autor: |
Beal J; Raytheon BBN Technologies, Cambridge, MA, USA. jakebeal@ieee.org., Farny NG; Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA, USA. nfarny@wpi.edu., Haddock-Angelli T; iGEM Foundation, Cambridge, MA, USA. traci@igem.org., Selvarajah V; iGEM Foundation, Cambridge, MA, USA., Baldwin GS; Department of Life Sciences and IC-Centre for Synthetic Biology, Imperial College London, London, UK. g.baldwin@imperial.ac.uk., Buckley-Taylor R; Department of Life Sciences and IC-Centre for Synthetic Biology, Imperial College London, London, UK., Gershater M; Synthace, London, UK. m.gershater@synthace.com., Kiga D; Faculty of Science and Engineering, School of Advanced Science and Engineering, Waseda University, Tokyo, Japan., Marken J; Department of Bioengineering, California Institute of Technology, Pasadena, CA, USA., Sanchania V; Synthace, London, UK., Sison A; iGEM Foundation, Cambridge, MA, USA., Workman CT; DTU-Bioengineering, Technical University of Denmark, Kongens Lyngby, Denmark. cwor@dtu.dk. |
| Abstrakt: |
Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data. |
