Optimizing Ribozyme Reporters Using RNase J1 in E.coli Based Cell-Free Systems

Abstract

Synthetic ribozyme reporters are RNA regulatory elements that can be modified to control the expression of downstream reporter proteins such as green fluorescent protein (GFP) in response to the binding of a target ligand. Ligand binding can activate or inhibit ribozyme self-cleavage, regulating the release of a mRNA segment that encodes for a fluorescent protein. RNase J1, a 5’-to-3’ endonuclease from Gram-positive bacteria, enables improved degradation of this protein in E. coli, unlike the native RNase E, which inefficiently degrades the transcript due to a 5’-hydroxyl group. We introduced RNase J1 into E. coli-based cell-free lysates and tested its effect on reporter expression. In a one-pot solution, RNase J1 decreased GFP expression in cell free reactions with the ribozyme reporters irrespective of ligand presence, however it did not decrease the expression of Pbab GFP, the control reporter tested. However, when testing a two-step reaction with titrated crude lysate containing RNaseJ1, we achieved expected performance in which RNaseJ1 decreased the fluorescent signal with the addition of a ligand. Further testing and optimization are needed to confirm that the decrease in fluorescent signal is both significant and reproducible, followed by additional optimization to increase the magnitude of that decrease.