Protein coding sequences/Reporters/Overview

Reporter protein coding sequences encode proteins whose presence in the cell or organism is readily observed. For example, fluorescent proteins cause a cell to fluoresce when excited with light of a particular wavelength, luciferases cause a cell to catalyze a reaction that produces light, and enzymes such as β-galactosidase convert a substrate to a colored product. Reporters are frequently used to quantify the strength or activity of upstream gene expression parts such as promoters and ribosome binding sites. Reporters, when fused in frame to other protein coding sequences, can also be used to identify where a protein is located in a cell or organism.

There are several different ways to measure or quantify a reporter depending on the particular reporter and what kind of characterization data is desired. Fluorescence can be readily quantified using a microscope, plate reader or flow cytometer equipped to excite the fluorescent protein with the appropriate wavelength of light. Moreover, since since several different fluorescent proteins are available, multiple gene expression measurements can be made in parallel. Luminescence can be readily quantified using a plate reader or luminescence counter. Luciferases are especially well-suited for measuring low levels of gene expression because cells tend to have little to no background luminescence in the absence of a luciferase. Enzymes that produce colored substrates can be quantified using spectrophotometers or other instruments that can take absorbance measurements including plate readers. Like luciferases, enzymes like β-galactosidase are good for measuring low levels of gene expression because they tend to amplify low signals. Generally speaking, microscopy is useful for obtaining both spatial and temporal information on reporter activity, particularly at the single cell level. Flow cytometers are best suited for measuring the distribution in reporter activity across a large population of cells. Plate readers are generally best for taking population average measurements of many different samples over time.