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

Antonia Lopreside,Elisa Michelini,Xinyi Wan,Aldo Roda,Baojun Wang

doi:10.1021/acs.analchem.9b04444

Antonia Lopreside, Elisa Michelini + Show 3 more

Open Access

https://doi.org/10.1021/acs.analchem.9b04444

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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 (gfpmut3, deGFP, mCherry, mScarlet-I), colorimetric (lacZ), and bioluminescent (luxCDABE from Aliivibrio fischeri and Photorhabdus luminescens, NanoLuc) 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, NanoLuc and deGFP 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.

Highlights

Whole-cell biosensors are cells that detect and report a target or condition of interest.[1−4] Due to being renewable, environmental friendly, and cost-effective, they have drawn increasing attention as viable alternatives to electronic or chemical sensors over the last three decades.[2,3]
Here we characterized and compared three widely used reporter categories, i.e., fluorescent, colorimetric, and bioluminescent reporters, under two representative biosensors of mercury- and quorum-sensing molecule within two different sensor settings, i.e., whole-cell and cell-free contexts. We investigated their properties in terms of contributions to analytical performance and key sensing features including limit of detection (LOD), input and output dynamic ranges, response time, and output visibility
We provided a comprehensive profiling of eight different genetic reporters from three commonly used reporter categories within two representative sensor systems both in vivo and in vitro

Summary

Analytical Chemistry

Article hydrolysis is fast,[19] but many bacterial strains contain an intact lac operon, which will increase the background of the colorimetric output. Here we characterized and compared three widely used reporter categories, i.e., fluorescent, colorimetric, and bioluminescent reporters, under two representative biosensors of mercury- and quorum-sensing molecule within two different sensor settings, i.e., whole-cell and cell-free contexts We investigated their properties in terms of contributions to analytical performance and key sensing features including limit of detection (LOD), input and output dynamic ranges, response time, and output visibility. Both colorimetric reporter LacZ and bioluminescent reporter NanoLuc under the mercury sensor were monitored with lysed and nonlysed cells (see Experimental Section) Both reporters’ performance was improved in cell lysing conditions in terms of response time, LOD, output dynamic range, and output visibilities (Figure 2D,E, Figures S2−S4), suggesting that the cell membrane could limit diffusion and transport of the substrates. Short time incubation will be required to obtain the best LOD and output dynamic range (Figure 3C, Figure S4) due to background activity induced by the sensor’s leakiness, which is more sensitive toward amplified enzymatic reactions than fluorescent reporters (Figure S2)

To test the generality of the aforementioned reporters’

■ CONCLUSIONS

Author Contributions

■ ACKNOWLEDGMENTS

■ REFERENCES