Comprehensive Profiling of Diverse Genetic Reporters with Application to Whole-Cell and Cell-Free Biosensors

Abstract

Whole-cell and cell-free transcription-translation biosensors have recently become favorable alternatives to conventional detection methods, as they are cost-effective, environmental friendly, and easy to use. Importantly, the biological responses from the biosensors need to be converted into a physicochemical signal for easy detection, and a variety of genetic reporters have been employed for this purpose. Reporter gene selection is vital to a sensor performance and application success. However, it was largely based on trial and error with very few systematic side-by-side investigations reported. To address this bottleneck, here we compared eight reporters from three reporter categories, i.e., fluorescent (<i>gfpmut3</i>, <i>deGFP</i>, <i>mCherry</i>, <i>mScarlet-I</i>), colorimetric (<i>lacZ</i>), and bioluminescent (<i>luxCDABE</i> from <i>Aliivibrio fischeri</i> and <i>Photorhabdus luminescens</i>, <i>NanoLuc</i>) reporters, under the control of two representative biosensors for mercury- and quorum-sensing molecules. Both whole-cell and cell-free formats were investigated to assess key sensing features including limit of detection (LOD), input and output dynamic ranges, response time, and output visibility. For both whole-cell biosensors, the lowest detectable concentration of analytes and the fastest responses were achieved with NanoLuc. Notably, we developed, to date, the most sensitive whole-cell mercury biosensor using NanoLuc as reporter, with an LOD ≤ 50.0 fM HgCl₂ 30 min postinduction. For cell-free biosensors, overall, <i>NanoLuc</i> and <i>deGFP</i> led to shorter response time and lower LOD than the others. This comprehensive profile of diverse reporters in a single setting provides a new important benchmark for reporter selection, aiding the rapid development of whole-cell and cell-free biosensors for various applications in the environment and health.