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

Abstract

Biosensors are biomolecular devices that detect ligands and are used in applications such as point-of-care screening. Ribozymes, RNA regulatory elements, can be introduced into biosensor designs to control the expression of reporter proteins like green fluorescent protein (GFP) in response to target binding. These systems are easily manipulated in cell-free protein expression (CFPE) systems, because they are not constrained by living cells. However, commercial CFPE kits are expensive, which hinders their accessibility, especially in low-income settings. Our lab previously developed a low-cost Rosetta 2(DE3)pLysS E. coli lysate for CFPE systems. We tested our system by expressing a naturally occurring glmS ribozyme reporter, coupled with the RNase J1 riboexonuclease to enhance degradation signals and improve ligand specificity for negative-signal ribozyme reporters. This study builds on that system by developing our own in-house energy mix. We are developing and testing a creatine phosphate creatine kinase regenerative system and comparing its performance to an existing PANOX energy mix that has a PEP-to-pyruvate ATP regenerative system. We also aim to produce the RNase J1 riboexonuclease to further evaluate its benefits in ribozyme-reporting diagnostics. A SUMO-His tagged RNase J1 plasmid was successfully constructed using Gibson Assembly and then transformed into BL21 cells. We aim to express and purify RNase J1 and utilize it in our ribozyme reporter system. Ultimately, our research aims to develop a more cost-effective and accessible cell-free biosensing platform for reliable ribozyme-based sensors.