Assessment of Colorimetric Reporter Enzymes in the PURE System

Colorimetric
reporter
enzymes are useful for generating eye-readable
biosensor readouts that do not require a device to interpret, an attractive
property for applications in remote or developing parts of the world.
The use of cell-free gene expression further facilitates such applications via amenability to lyophilization and incorporation into
materials like paper. Currently, detection of multiple analytes simultaneously
with these systems requires multiple reactions or a device. Here we
evaluate seven enzymes and 15 corresponding substrates for functionality
in a particular cell-free expression system known as PURE. We report
eight enzyme/substrate pairs spanning four enzymes that are compatible
with PURE. Of the four enzymes, three pairings exhibit no cross-reactivity.
We finally show that at least one pairing can be used to create a
third color when both are present, highlighting the potential use
of these reporters for multiplex sensing.