The current COVID-19 pandemic presented an imperative lesson on the importance of on-demand diagnostics to detect novel viruses, which led more people to notice the practical potential of on-demand diagnostics to track other health indicators. However, a major pitfall for the current on-demand diagnostics is sacrificing accuracy for cost. The cell-free protein synthesis (CFPS) system can alleviate this concern, as it is a tunable, lyophilized, low-cost system designed to focus on carrying out the protein synthesis exclusively without the energy costs of cell growth. The users can create complex gene circuits to operate the complex protein synthesis cascades responding to the analyte of interest. Here, we have added a new gene circuit to the cost-effective on-demand CFPS diagnostics toolbox, allowing for higher accuracy. This circuit consists of synthesizing a 1) robust non-native sigma factor that binds specifically to its cognate promoter, 2) sigma factor synthesis of a more robust bacteriophage RNA polymerase (RNAP), and 3) synthesis of a bright fluorescent green protein. Using this non-native sigma factor allow the user more modulation of parts using a cognate promoter, compared to a bacteriophage RNAP, and for a large reduction in background signal, compared to a native <i>Escherichia coli </i>sigma factors. In addition, the systemic optimization of endogenous <i>E. coli </i>RNAP activity was studied in regard to the types of DNA templates, cell-extract preparation, and CFPS reagents. This CFPS-based gene circuit will allow for low-cost on-demand diagnostics without sacrificing accuracy.

Enhancing on-demand diagnostics with a non-native sigma factor-based cell-free protein synthesis gene circuit

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.