Biology subjects

Copeland, C. E.

Publications and source records attributed to Copeland, C. E..

3 recordsLinked to original sources

bioRxiv2023

Expanding the Cell-Free Reporter Protein Toolbox by Employing a Split mNeonGreen System to Reduce Protein Synthesis Workload

The cell-free system offers potential advantages in biosensor applications, but their limited substrate supply poses a challenge in balancing enough sensing resources to detect low limits of analyte while providing a robust output signal. In this study, we harnessed split versions of fluorescent proteins, particularly split super-folder green fluorescent protein and mNeonGreen, to improve energy efficiency and enhance detection limit in the cell-free system. A comparative analysis of the expression of 1-10 and eleventh segments of beta strands in both whole-cell and cell-free platforms revealed distinct fluorescence patterns. Moreover, integrating SynZip peptide linkers substantially improved complementation, achieving a fluorescence intensity reaching 73.6% of the full-length protein and a 4.8-fold increase in expression compared to the split system without the SynZip peptide linkers. The split protein reporter system can enable energy-efficient sensing of low analyte levels in the cell-free system, broadening the toolbox of cell-free biosensor repertoire.

synthetic biology↗

bioRxiv2023

Suitability Evaluation of Toehold Switch and EXPAR for Cell-Free MicroRNA Biosensor Development

The development of a robust and cost-effective sensing platform for microRNA (miRNA) is of paramount importance in detecting and monitoring various diseases. Current miRNA detection methods are marred by low accuracy, high cost, and instability. The toehold switch riboregulator has shown promising results in detecting viral RNAs integrated with the cell-free system (CFS). This study aimed to leverage the toehold switch technology to detect miRNA in the CFS and to incorporate the exponential amplification reaction (EXPAR) to bring the detection to clinically relevant levels. We assessed various EXPAR DNA templates under different temperatures and additives to enhance the accuracy of the sensing platform. Furthermore, different structures of toehold switches were tested with either high-concentration synthetic miRNA or EXPAR product to assess sensitivity. Herein, we elucidated the mechanisms of the toehold switch and EXPAR, presented the findings of these optimizations, and discussed the potential benefits and drawbacks of their combined use. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/540462v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1902d64org.highwire.dtl.DTLVardef@94313forg.highwire.dtl.DTLVardef@12d59ddorg.highwire.dtl.DTLVardef@66ecfc_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

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

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 biosensors dynamic range must be increased in the system to achieve the 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 false positives proportion.

synthetic biology↗