When Wavelengths Collide: Bias in Cell Abundance Measurements Due to Expressed Fluorescent Proteins

The abundance of bacteria in liquid culture is commonly inferred by measuring optical density (OD) at 600 nm. In doing so, it is commonly assumed that the dominant source of the attenuation of incident light is scattering by cells. If cells express fluorescent proteins that absorb light at the wavelength used for measuring OD, then this absorption can also attenuate incident light. Red fluorescent proteins (RFP) can strongly absorb light at 600 nm. Increasing RFP expression can falsely inflate apparent cell density and lead to underestimations of mean per-cell fluorescence by up to 10%. A literature search shows that this error is prevalent through the synthetic biology community.

We will present data showing that this underestimation of mean per-cell fluorescence is linear with the amount of expressed RFP. We will also present data showing that measuring OD at 700 nm would allow estimation of cell abundance unaffected by the presence of nearly all fluorescent proteins. 

As the practice of engineering cells becomes ever more precise there is a corresponding need for better characterization of the impact of genetic elements on gene expression. We believe that raising awareness of this issue within the synthetic biology community, and promoting adoption of the simple change that we propose, will improve the quality of data generated throughout this community.