"Characterizing a New Fluorescent Protein for a Low Limit of Detection " by Caroline E. Copeland, Jeehye Kim et al.

Faculty Publications

Title

Characterizing a New Fluorescent Protein for a Low Limit of Detection Sensing in the Cell-Free System

Authors

Caroline E. Copeland, Department of Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, Louisiana 70803, United States.
Jeehye Kim, Department of Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, Louisiana 70803, United States.
Pearce L. Copeland, Department of Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, Louisiana 70803, United States.
Chloe J. Heitmeier, Department of Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, Louisiana 70803, United States.
Yong-Chan Kwon, Department of Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, Louisiana 70803, United States.

Document Type

Article

Publication Date

8-19-2022

Abstract

Cell-free protein synthesis-based biosensors have been developed as highly accurate, low-cost biosensors. However, since most biomarkers exist at low concentrations in various types of biopsies, the biosensor's dynamic range must be increased in the system to achieve low limits of detection necessary while deciphering from higher background signals. Many attempts to increase the dynamic range have relied on amplifying the input signal from the analyte, which can lead to complications of false positives. In this study, we aimed to increase the protein synthesis capability of the cell-free protein synthesis system and the output signal of the reporter protein to achieve a lower limit of detection. We utilized a new fluorescent protein, mNeonGreen, which produces a higher output than those commonly used in cell-free biosensors. Optimizations of DNA sequence and the subsequent cell-free protein synthesis reaction conditions allowed characterizing protein expression variability by given DNA template types, reaction environment, and storage additives that cause the greatest time constraint on designing the cell-free biosensor. Finally, we characterized the fluorescence kinetics of mNeonGreen compared to the commonly used reporter protein, superfolder green fluorescent protein. We expect that this finely tuned cell-free protein synthesis platform with the new reporter protein can be used with sophisticated synthetic gene circuitry networks to increase the dynamic range of a cell-free biosensor to reach lower detection limits and reduce the false-positive proportion.

Publication Source (Journal or Book title)

ACS synthetic biology

First Page

2800

Last Page

2810

Recommended Citation

Copeland, C. E., Kim, J., Copeland, P. L., Heitmeier, C. J., & Kwon, Y. (2022). Characterizing a New Fluorescent Protein for a Low Limit of Detection Sensing in the Cell-Free System. ACS synthetic biology, 11 (8), 2800-2810. https://doi.org/10.1021/acssynbio.2c00180

Link to Full Text

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