Optimization of an in-house bacterial cell-free expression system to evaluate the design of toehold switch sensors for selected cervical precancer miRNA biomarkers

Abstract

Cervical cancer is a significant health burden for women across the globe. However, over 94% of all cervical deaths occured in low and middle income countries (LMICs) in 2022 alone. This reflects a broader trend of a lack of access to quality treatment options for cervical cancer due to socioeconomic barriers including limited trained professionals, financial resources, and access to proper screening. We are partnering with UMaryland iGEM, an undergradutate-led synthetic biology research team, to assist in the development of a low-cost, point-of-care screening device for detecting commonly upregulated miRNAs in cervical precancer patients to address this issue. The functionality of this device relies on a cell-free expression kit, referred to as a lysate, in order to properly transcribe the desired toehold switches in the paper-based assay and translate the fluorescent protein output. However, common commercial cell-free lysates are expensive which increases the cost of producing our device – ultimately hindering its accessibility in LMICs. To address this problem, we propose a method for producing and optimizing our own in-house cell-free lysate. In our method, we compare three lysates – commercially available myTXTL, a lysate developed by the Aberdeen Proving Ground, and our own lysate – using varying ratios of energy mix, lysate, and T7p14 deGFP HP plasmid to evaluate the ideal composition of these components for a cell-free expression kit. We quantify these relationships using fluorescence in order to determine the efficacy of our lysate and the ideal ratio of components for a cell-free, paper-based assay.