Accessible, In-Lab Cell-Free Expression System for Biosensor Development

Abstract

Early detection of medical and environmental conditions is difficult, as external markers may develop long after cellular and molecular markers do. There is a need for point-of-care devices that can quickly and easily detect these markers in various samples. RNA-based fluorescent biosensors offer an easily customizable solution for detection and reporting. A major limiting factor in the deployment of biosensors is their reliance on living cells; cell-free protein expression (CFPE) systems can overcome this barrier. However, commercial CFPE kits are expensive, which hinders their overall use and deployability, especially in low-income settings. To address this pressing challenge, we developed our own Rosetta 2(DE3)pLysS E. coli cell-free protein expression system that can be produced at a lower cost. To evaluate its performance, we compared it to a commercially available kit with standardized fluorescent reporters. While our design underperformed commercially available lysate, it performed comparably to a lysate developed by the U.S. Army Combat Capabilities Development Command (DEVCOM). We aim to optimize our lysate and protocols to further increase performance and develop point-of-care devices for disease biomarkers of interest.